http://2009.igem.org/wiki/index.php?title=Special:Contributions/Bammarata89&feed=atom&limit=50&target=Bammarata89&year=&month=2009.igem.org - User contributions [en]2024-03-29T06:49:55ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:CornellTeam:Cornell2010-10-24T20:00:26Z<p>Bammarata89: </p>
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=Project Abstract=<br />
[[Image:CUTeam.JPG|right|500px]]</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T03:58:49Z<p>Bammarata89: </p>
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= Team Description =<br />
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|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
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= Advisors =<br />
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<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
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= Undergrads =<br />
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== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
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== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
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== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
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== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
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== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
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== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
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|}</div>Bammarata89http://2009.igem.org/Team:Cornell/PartsTeam:Cornell/Parts2009-10-22T03:58:07Z<p>Bammarata89: </p>
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=Parts=<br />
===Device===<br />
''Cadmium Sensor (BBa_K285104)''<br />
expresses GFP as a function of Cadmium Ion concentration. The first part of the GFP Cadmium Sensor is the CadA regulatory element which increases expression in response increasing Cadmium concentration. This is followed by spoVG a ribosome binding site for Bacillus Subtilis. The GFP coding sequence follows. Note that the GFP Cadmium Sensor will only function with the czrA regulon found in Bacillus Subtillis. <br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
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=== Individual Parts===<br />
''CadA Regulatory Region (BBa_K285101,[http://partsregistry.org/Part:BBa_K285101:Design])'' is the regulatory sequence upstream of the cadA gene (previously known as yvgW). This sequence is part of the czrA regulon and is regulated by the CzrA repressor which is derepressed in the presence of Cd2+.[1][2]<br />
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''copZA Regulatory Region (BBa_K285102,[http://partsregistry.org/Part:BBa_K285102:Design])'' is the regulatory sequence upstream of the copZ and copA genes. Activation of the copZ gene proceeds by copper mediated induction by the CueR protein which binds to this region.[2]<br />
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''mntH Regulatory Region (BBa_K285103,[http://partsregistry.org/Part:BBa_K285103:Design])''is upstream of the mntH gene is repressed by the MntR protein which is derepressed upon induction by Cd2+.[1][3]<br />
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=== References===<br />
[1]Moore CM, Gaballa A, Hui M, Ye RW, Helmann JD. Genetic and physiological responses of Bacillus subtilis to metal ion stress. Mol Microbiol. 2005 Jul;57(1):27-40.<br />
<br />
[2]Ahmed Gaballa & John D. Helmann Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems. Biometals. 2003 Dec;16(4):497-505. <br />
<br />
[3]Que Q, Helmann JD.Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins.Mol Microbiol. 2000 Mar;35(6):1454-68.</div>Bammarata89http://2009.igem.org/Team:Cornell/PartsTeam:Cornell/Parts2009-10-22T03:55:02Z<p>Bammarata89: </p>
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=Parts=<br />
===Device===<br />
''Cadmium Sensor (BBa_K285104)''<br />
expresses GFP as a function of Cadmium Ion concentration. The first part of the GFP Cadmium Sensor is the CadA regulatory element which increases expression in response increasing Cadmium concentration. This is followed by spoVG a ribosome binding site for Bacillus Subtilis. The GFP coding sequence follows. Note that the GFP Cadmium Sensor will only function with the czrA regulon found in Bacillus Subtillis. <br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
<br />
=== Individual Parts===<br />
''CadA Regulatory Region (BBa_K285101,[http://partsregistry.org/Part:BBa_K285101:Design])'' is the regulatory sequence upstream of the cadA gene (previously known as yvgW). This sequence is part of the czrA regulon and is regulated by the CzrA repressor which is derepressed in the presence of Cd2+.[1][2]<br />
<br />
''copZA Regulatory Region (BBa_K285102,[http://partsregistry.org/Part:BBa_K285102:Design])'' is the regulatory sequence upstream of the copZ and copA genes. Activation of the copZ gene proceeds by copper mediated induction by the CueR protein which binds to this region.[2]<br />
<br />
''mntH Regulatory Region (BBa_K285103,[http://partsregistry.org/Part:BBa_K285103:Design])''is upstream of the mntH gene is repressed by the MntR protein which is derepressed upon induction by Cd2+.[1][3]<br />
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=== References===<br />
[1]Moore CM, Gaballa A, Hui M, Ye RW, Helmann JD. Genetic and physiological responses of Bacillus subtilis to metal ion stress. Mol Microbiol. 2005 Jul;57(1):27-40.<br />
<br />
[2]Ahmed Gaballa & John D. Helmann Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems. Biometals. 2003 Dec;16(4):497-505. <br />
<br />
[3]Que Q, Helmann JD.Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins.Mol Microbiol. 2000 Mar;35(6):1454-68.</div>Bammarata89http://2009.igem.org/Team:Cornell/Project/DesignTeam:Cornell/Project/Design2009-10-22T03:46:18Z<p>Bammarata89: /* Proposed Design */</p>
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== Proposed Design ==<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
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Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions. [7]<br />
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==Cadmium Sensor Module 1==<br />
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Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
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[[Image:Module 1 Inactive.GIF|600px|center]]<br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
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==Cadmium Sensor Module 2==<br />
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Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
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[[Image:Module 2 Inactive.GIF|600px|center]]<br />
[[Image:Module 2 Active.GIF|600px|center]]<br />
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By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
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=== References ===<br />
[7]Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
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Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40</div>Bammarata89http://2009.igem.org/Team:Cornell/NotebookTeam:Cornell/Notebook2009-10-22T03:41:27Z<p>Bammarata89: </p>
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6/26/09<br />
Alyssa and Matt—made chloramphenicol, kanamycin, and tetracycline antibiotic stock solutions for use in agar plates. See “Protocols” folder for antibiotic making protocol.<br />
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Antibiotic Volume (mL): Stock (mg/mL): Amnt use (g): Solvent<br />
chloramphenicol 15 34(200x) 0.510 EtOH<br />
kanamycin 15 10(200x) 0.150 H20<br />
tetracycline 50 5(200x) 0.250 EtOH<br />
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Comments: <br />
Please note that tetracycline does NOT fully dissolve in 100% EtOH—will see a yellow powder in a clear solution.<br />
For all stock solutions of antibiotics dissolved in H20: “sterilize by filtration through a 0.22um filter (Sambrook and Russel)”<br />
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Xing—PCR cloning of GFP+ into Biobrick standard A<br />
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6/29/09<br />
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Malinka and Ben—Spread antibiotics on agar plates for ccdb amplification. Created a table for the antibiotics we were going to use and then top spread antibiotics onto agar plates. <br />
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Comments:<br />
The color code for the antibiotics: <br />
Amp – Black<br />
Chlor – Red<br />
Kan – Green<br />
Tet – Blue<br />
Table they came up with is Ben’s notebook entry for the day. Results are summarized as the following: <br />
--pSB1AK3 had colonies on each plate<br />
water plates had no colonies, except for amp plates<br />
pSB1AC3 and pSB1AT3 had no colonies, except for on the amp plates<br />
Xing Agarose Gel Electrophoresis Analysis of GFP+ PCR Product to analyze products of PCR of GFP+<br />
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Lanes 1 2 3 4 5 6 7 8<br />
PFx 1ng 5λ <br />
PFx 10ng 5λ <br />
PFx 100ng 5λ <br />
Pfu 1ng 5λ <br />
Pfu 10ng 5λ <br />
Pfu 100ng 5λ <br />
pGFP+ 1 5λ <br />
100bp ladder Fermentas 5λ<br />
6X Loading dye 2λ 2λ 2λ 2λ 2λ 2λ 2λ <br />
Total Volume 7λ 7λ 7λ 7λ 7λ 7λ 7λ 5λ<br />
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Comments:<br />
The rxns using Pfx display product at the expected mobility ~750bp with increasing yields following increasing amount of template plasmid used. The rxns treated with Pfu displayed no product.<br />
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6/30/09<br />
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Xing—PCR Amplification of yvgY promoter & GFP+. The goal was to PCR Amplify yvgY promoter from chromosome into BioBrick standard and repeat GFP+ amplification since we used 10X more primer in the previous experiment than we should have.<br />
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Malinka and Ben—extracted iGEM backbone plasmids from kit (wells 11c for pSB1AC3, 13e for pSB1AK3, and 15c for pSB1AT3) and transformed them. Also grew B. subtilis strains to make frozen permanents. <br />
Comments:<br />
Ben's plates (for pSB1AK3) had various numbers of colonies<br />
Malinka’s plates (for pSB1AC3 and pSB1AT3)<br />
Nothing grew on the 50 ul of only Tet or ChlorzNothing grew in the 50 ul or 250 ul of the Tet and amp and the Chlor and Amp<br />
No colonies on water plates<br />
Some cells grew on the only amp plates. Thus amp not very effective, and there was a large “background”<br />
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7/1 <br />
Xing; Agarose Gel Electrophoresis of GFP+/yvgY promoter PCR Products<br />
GFP+ bands display expected mobility, with greater yield in the 1ng rxn than the previous rxn. No apparent product in rxns amplified by Pfu. yvgY shows up faintly for rxns using Pfx and Pfu with expected mobility <100bp. <br />
Alyssa and Matt: Growth Curve Characterization of B Subtilis at Different Culture Scales To characterize the growth curves of B Subtilis in both ml scale tubes and in microtiter plates by measuring optical density at set intervals during growth. (By finding when the strains enter logarithmic growth phase, we can better design our B galactosidase assay protocol.) Calculations assume midlog phase.<br />
Ben: Inoculating pSB1AK3 & re-plating pSB1AC3 and pSB1AT3. Results: Inoculating pSB1AK3<br />
All tubes contain cells…SUCCESS! Re-plating pSB1AC3 and pSB1AT3<br />
None of the water plates grew cells, except for the tet lower concentration plate<br />
The supposed plasmid containing cells also did not grow, except for some background on the lower concentration tet plate.<br />
Malinka: Replating plasmids and making frozen permanents Replate the pSB1AC3 and pSB1AT3 and do frozen permanents<br />
Results: Plasmids didn’t grow, lawn on water<br />
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7/2 <br />
Xing: Gel Electrophoresis of yvgY promoter PCR rxn with primers<br />
Compare yvgY PCR rxns with primers to confirm or refute presence of product<br />
Primers do not show up on gel, there is a band below 100bp for the PCR rxns. The expected product size is ~120 bp. The <100 band could be due to primer dimerization. Run a primer titration down to 50nM primer to check next time.<br />
Ben: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain. Results: Re-transforming pSB1AC3 and pSB1AT3-No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates). plating ccdB-resistant strain -2 lawns formed<br />
Malinka: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain<br />
Results: -No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates) plating ccdB-resistant strain-2 lawns formed<br />
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7/3 <br />
Xing: Preparation of Chemically Competent E.Coli. Part II<br />
Xing: Gel Analysis PCR of yvgY and yvgW promoters primer titration; Run agarose gel electrophoresis to analyze PCR rxns yvgY rxns display the same band <100bp but appears less intense for .1ng/uL primer rxn and even less for .05ng/uL primer rxn, supporting the hypothesis that these are primer dimerization products. yvgW does not display any products. Possibility of broken chromosomal DNA? Extract Bacillus DNA next time.<br />
Xing: PCR Amplification of yvgY and yvgW promoter PCR amplify yvgY and yvgW promoter into BB standard and titrate primers to see if proper product forms.<br />
Xing: Prepare Kanamycin and Tetracycline Plates<br />
Ben: Preparation of Chemically Competent E.Coli.<br />
Malinka: Prepare Chloramphenicol and Ampicillin Plates, and grow competent cells<br />
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7/6<br />
Ben and Malinka: Checking competency of cells/ if plates are working (by Malinka). Bad results, redo experiment<br />
7/7<br />
Ben: Re-re-transforming cells (LOL). Transform (hopefully) pSB1AC3 and pSB1AT3 in ccdB-resistant and DH5a strains.<br />
--Results: -Tet plates (including water and both pSB1AT3 plates) contained lawns, reason unkown<br />
-Chlor plates came out as predicted, nothing on water plates, dozens of colonies on 250uL plate, <10 on 50uL plate, but colonies were not red still need to view them under UV light<br />
-water controls were clean, except for Tet, as said above<br />
-pUC19 1:10 dilution had <10 colonies, 1:100 had none visible replating at 1:1 to check competent cells’<br />
Malinka: Replating plasmid backbones-- Playing pSB1AK3 and pSB1AC3 on new antibiotic plates<br />
--Results: pSB1AT3 (ccdb) – Proportional amounts of single colonies had grown on my 50 and 250 ul plates. However a similar amount of colonies had grown on the water too. <br />
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pSB1AT3 (DH5a) – Ben had lawns on all his Tet plates.<br />
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pSB1AC3 (ccdb) – I had nothing on all 3 of my chlor plate.<br />
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pSB1AC3 (DH5a) – Proportional amounts of single colonies had grown on his 50 and 250 ul plates. Nothing had grown on his water. The cells weren’t red like we expected them to be but that was probably because they were too small.<br />
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Water (1:1000) (ccdb and DH5a) – We had lots of single colonies on both our plates. They looked similar.<br />
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pUC19 (ccdb) - <br />
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pUC19 (DH5a) - <br />
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7/8<br />
Xing: PCR yvgY promoter & yvgW temperature using a temperature gradient<br />
Xing: Preparation of mntR frozen permanents<br />
Ben: Innoculating pSB1AC3 and pSB1AT3 (rfp versions) to see if we have transformed backbones. <br />
--Results: Chlor plasmids had growth, reddish in tube!-Tet did not grow, as expected<br />
Malinka: culture backbone from the plates done yesterday<br />
--Results: All of Ben’s pSB1AC3s grew and turned red. Only 1 of Malinka’s pSB1AT3s grew<br />
Alyssa: Preparing B. subtilis cultures exposed to ZnCl2 for practice LacZ Assay-- To try out the B-Gal assay protocol mentioned on page 12 of Helmann’s “Genetic and physiological responses of B. subtilis to metal ion stress” paper<br />
--Results: Whoa! Heavy metals/ growth retardation in action! According to the last OD600 readings, ZnCl2 seems to significantly (and negatively) affect the growth rates of the B. subtilis strains exposed to the highest concentrations of metal solution.<br />
<br />
7/9 <br />
Xing: Agarose Gel Electrophoresis of PCR yvgY yvgW temperature gradient to analyze PCR products-- yvgY rxn still forms primer dimmers, though concentration seems to decrease with increasing temperature. No products for yvgW rxn.<br />
Matt and Alyssa: Designed Zinc growth curve plate<br />
Ben: Mini-prep pSB1AC3 (rfp) and pSB1AK3 (ccdB)<br />
7/10<br />
Xing: Agarose Gel Electrophoresis of Temperature Gradient to analyze PCR products<br />
Xing: PCR temperature gradient of yvgY & yvgW-- PCR yvgY & yvgW promoters with positive controls.<br />
7/13<br />
Malinka: Digesting the plasmid backbones PSB1AC3 and PSB1AK3<br />
<br />
7/14<br />
Xing: Gel electrophoresis of varying [Mg2+] PCR<br />
Xing: PCR Amplification of yvgY & yvgW w/ varying [Mg2+]-- Vary [Mg2+] in PCR rxn to see if products form<br />
Malinka: ran gels for backbones<br />
7/15<br />
Matt: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
Malinka: Digestion - Removing inserts from the plasmid backbones PSB1AC3, PSB1AK3, PSB1AT3<br />
--Results: <br />
<br />
7/16<br />
Ben: DNA electrophoresis of doubly digested (X & S) backbones and GFP+<br />
7/17<br />
Xing: PNK treatment and annealing of complementary primers-- PNK treat primers to phosphorylate ends for ligation/anneal complementary primers<br />
Alyssa: CdCl2 soln growth curve plate design for use in TECAN machine<br />
7/20<br />
Ben: Check of pSB1AK3 ccdB-gene effectiveness through transformation in DH5a<br />
--Results: -waters were blank-puc19 had a few cells -sSB1AK3 plates both transformed, meaning the ccdB gene is broken, since the cells should have died<br />
Malinka: Dephosphoralating and purifying backbone plasmids PSB1AC3, PSB1AK3, PSB1AT3<br />
7/21<br />
Matt and Alyssa: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
Ben: Ligation of promoters and GFP+<br />
Malinka: Creating GFP and promoter biobricks—yvgW and yvgY<br />
--Results: Based on yesterdays Tet plate results, we found that the Tet plates work. But the ccdb psb1AK3 does not work so we have only pSB1AC3 and pSB1AT3 as backbones.<br />
7/22<br />
Ben: Transformation of ligations yvgW and GFP+<br />
Malinka: Redigestion - Removing inserts from the plasmid backbones<br />
Results: Plated Ligated Biobricks <br />
<br />
Controls:<br />
The pUC19 on amp showed reasonable growth and the water on amp had nothing which meant that there was no background on the amp plates. As we still got good growth for pUC19, this showed that DH5 cells were alive and competent. The water on the Chlor plater had no growth. <br />
<br />
The no insert and no enzyme ligation mixtures showed no growth.<br />
<br />
yvgW and yvgY:<br />
There was little growth on the 2:1 ratio plates for yvgW and yvgY (maybe 5 colonies). There was more growth on the 6:1 ratio plates (maybe 15 colonies) – we plan to use 6:1 ratio from now on.<br />
<br />
GFP:<br />
There was no growth on any of the GFP biobrick plates (2:1, 4:1 or 6:1).<br />
<br />
Undigested plasmid (pSB1AC3) would have shown up as red cells on the plate. However from previous experience, we knew that 24 hours of incubation wasn't enough for the cells to have grown large enough or long enough to be visibly red. So we weren't sure, of the plates with growth in them, which cells actually had been transformed with our biobricks as opposed to the undigested plasmid. <br />
<br />
7/23<br />
Ben: Transformation of pSB1K3 and pSB1T3 backbones<br />
--Results: -Kan plates produced colonies (slightly red) -waters had nothing (except tet plates bad plates) -pUC19 had colonies -tet plates all had lawns bad plates<br />
Malinka: To run gels on the newly digested plasmid backbones and cut GFP to make sure we had got them right. To dephosphorylate the cut backbones and then purify them so that we had enough vector to ligate with our inserts later.<br />
--Results: The gels looked good for Chlor, Tet and Kan. I was even able to see a the insert for Kan very low in the gel (it was very faint) – exactly where the iGEM gel showed it to be.<br />
<br />
I got nothing for the cut and uncut GFP. Ben had used uncut GFP from the same tube a few days ago and got results but he used 1 ul which is (600-700 ng) so perhaps I had too little. We need to do this again with more cut and uncut GFP.<br />
<br />
<br />
Xing – To culture tubes with colonies from our transformed biobricks (with promoters). Also to culture 1 tube with pSB1AC3 (with RFP) because we were going to use RFP (and GFP) for our project. <br />
<br />
Ben and Matt – To make new Chlor and Tet plates and transform cells with new backbone plasmid from the kit (pSB1T3 & pSB1K3) in case our next backbone doesn't work. <br />
7/24<br />
Cadmium and Zinc Beta Galactosidase Assay in tubes: To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion. Couldn’t access spectrometer when the assay was complete, so tubes were stopped when they showed a clear yellow color and left to sit out over the weekend. On Monday all remaining tubes were stopped and OD420, OD550, and OD600 were measured in the spec.<br />
Malinka: Test--Digesting the pSB1AC3 and pSB1AT3 using 15 minute method and digesting purified GFP+<br />
--Results: The 15 minute digest of pSB1AC3 and pSB1AT3 showed the same results as the digests of the same plasmids done overnight.<br />
<br />
We saw the uncut GFP but did not see the cut GFP. We think it’s because the GFP+ wasn’t purified.<br />
<br />
<br />
<br />
7/24<br />
Xing: Purification of GFP+ PCR Products<br />
<br />
7/27<br />
Xing; Purification of Additional GFP+ PCR Products<br />
Alyssa and Malinka: Digesting GFP+ and running gels on it-- To test pure cut GFP+ digested for 6 hours, cut and then test GFP+ digested for 15 minutes, cut and then test new backbones, and cut and then test biobrick parts with promoters.<br />
7/29<br />
Ben: Mini-prep of GFP (E0040), CFP (E0020), and YFP (E0030)<br />
Alyssa: designed primers to extract constitutive mrgA promoter from Helmann strain of B. sub.<br />
7/30<br />
Alyssa and Xing: Digest pSB1K3 & ligate GFP+ part onto digest--Digestion of assembly vector pSB1K3, dephosphorylation & ligation of GFP+ onto assembly vector. <br />
Ben: Digestion of GFP CFP and YFP-- Appropriate bands located in all wells: perfect!<br />
<br />
<br />
<br />
7/31<br />
Xing: Transformation of GFP+ Ligation and selection with Kanamycin<br />
Xing: Transformation of pSB1AC3 using Amp selection--1 Colony for 50uL pSB1AC3<br />
~ 10 Colonies for 250 uL pSB1AC3, no colonies for H2O, pUC19 250 uL 0-5 colonies.<br />
Alyssa and Ben: Ligation of assembly vector PSBK3 to GFP+ to create GFP+ biobrick parts<br />
<br />
8/3<br />
Xing: Digest & gel analysis of yvgW, yvgY, GFP+ BB parts<br />
- No GFP+ parts, incomplete digestion for yvgW, yvgY parts.<br />
Malinka: Mini-prepped GFP+ biobricks and pSB1AC3<br />
8/4 Xing: Digest & gel analysis of yvgW, yvgY BB parts<br />
- Mobility as expected for for yvgW 2:1 1 and yvgY 6:1 3<br />
Malinka: Transformed and plated pSB1AT3 (rfp and ccdb) from the kit.<br />
<br />
8/5 Xing: Testing effectiveness of tetracycline plates<br />
- Lawn formed on all tet plates<br />
Malinka: Cultured pSB1AT3 (DH5) cells, and yvgY (on pSB1AC3), yvgW (on pSB1AC3) and GFP+ (on pSB1AK3) biobricks.<br />
Matt: Previous Tetracyclin plates have failed to kill strains without Tet resistance. Matt made another attempt to create working Tetracyclin using a new improved stock of 100X Tet. A new Tetracycline stock was created. Previous stocks were made using 100% Ethanol under the assumption that we were working with pure Tetracyline. However, our solid stock is actually Tetracyline Hydrochloride. This compound has difficulty suspending in pure Ethanol. Using a 70% Ethanol 30% water solvent the Tetracyline Hydrochloride dissolved almost instantly. Previously no precautions were made to counter Tetracyline’s light sensitivity. This time we wrapped the stock in tin foil and were careful to avoid too much light exposure.<br />
<br />
<br />
Digest & gel analysis of yvgW 2:1 1, yvgY 6:1 3, GFP+ 2:1 50 BB parts<br />
- yvgW 2:1 1 sample with expected mobility, yvgY 6:1 3 sample incompletely digested, GFP+ 2:1 50 sample not as expected<br />
<br />
8/6 Matt characterize Tet’s effect on cells when top spreading and in liquid culture. All of the 0x, .1x, and 1x Tet tubes grew to saturation while neither of the 10x Tet tubes grew. Further testing is necessary to determine if a concentration can be found where Tet resistant strains grow but strains without Tet resistance do not grow. All of the plates grew. However, 1X Tet with Tet resistant strain PSB1AT3 in DH5alpha did not grow to a lawn but showed many (>100) small single colonies.<br />
<br />
8/7 Matt: Previously we determined that both Tet resistant and unresistant cells grow in 1x Tet liquid culture but die in 10x liquid culture. Our purpose is to determine a Tet concentration between these two extremes where Tet resistant culture lives and Tet unresistant culture does not. Cultures were cloudy for pSB1AT3 & pUC19: 0X tet, 2X tet. Cultures were clear for pSB1AT3 & pUC 19: 4X tet, 6X tet, 8X tet. OD600 for pSB1AT3 2X tet(10X dilution): 0.24 OD600 for pUC19 2X tet(10X dilution): 0.18 Not a significant OD600 difference to select from. Test range from 2X-4X tet next.<br />
<br />
<br />
<br />
8/10 Xing: Transformation of BB_spoVG RBS & BB_mrgApromoterrbs<br />
Failed transformation<br />
Alyssa: Made Excel template for ligation calculations. Phosphorylated, annealed, and ligated purchased oligonucleotides:<br />
mrgA, mntH, spoVG1, spoVG2<br />
<br />
Malinka: Phosphorylated and annealed mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS<br />
Matt: Previously we determined that both Tet resistant and unresistant cells grow in 1x Tet liquid culture but die in 10x liquid culture. Our purpose is to determine a Tet concentration between these two extremes where Tet resistant culture lives and Tet unresistant culture does not. Using strains: PSB1AT3 in DH5alpha and PUC19 in DH5alpha at concentration of 0X, 2.5X, 3X, 3.5x, and 4X Tet<br />
(1X Tet= 50ug/ml, stock is at 100X) Culture in 5ml of LB, inoculate from picking plates. At 2.5X PSB1At3 DH5alpha grew but PUC19 DH5alpha did not. Above this concentration nothing grew.<br />
<br />
<br />
<br />
8/11 Malinka: : Transformed and plated mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS (all on pSB1AC3).<br />
Alyssa: Ligated and transformed oligos of mntH promoter and mrgA promoter. Transformed and plated RBS and mnth promoter biobricks in DH5 cells<br />
<br />
<br />
<br />
8/12 Xing: Test transformation protocol and competency of cells <br />
Cells competent and transformation worked with pUC19<br />
Alyssa: CdCl2 growth curve plate design<br />
<br />
<br />
8/13 Xing: Tranformation of mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
Successful transformation<br />
Ben: Inoculated spoVG transformants for mini-prepping<br />
Malinka: Cultured amyE parts and made amp plates.<br />
Alyssa:<br />
Redid CdCl2 growth curves redo the CdCl2 growth curve experiment from 7/18, which resulted in biphasic growth curves and growth of mntR when exposed to Cd. (This should not have happened due to its extreme Cd sensitivity.)<br />
--Results:<br />
No errors <br />
111 runs<br />
Biphasic growth curves for all strains, though not as pronounced as 1st growth curve exp.<br />
mntR shows more growth than WT, even at 5uM Cd <br />
<br />
<br />
<br />
8/14 Xing: Inoculate cultures from transformant plates for mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
PAGE purification of mrgApromrbs<br />
Alyssa:<br />
Made new 1000X Tet stock and measured concentration of PSB1K3-- to make a more potent Tet stock soln. and measure concentration of a biobrick backbone for calculation purposes in other experiments and procedures (such as ligation).<br />
Analyzed data from 8/13 growth curves on Excel<br />
Cut and dephosphorylated psB1K3 backbone<br />
<br />
<br />
8/15 Xing: Digest and gel analysis of spoVG, BBa_K143001, BBa_K143002<br />
spoVG migrates with expected mobility, BBa_K143001 & 2 do not migrate as expected<br />
<br />
Miniprep mntH, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
8/19 Alyssa: Digestion of mntH, mnrA promoter, mrgA + mrgA rbs, and mrgA rbs<br />
Matt: digested Biobricks in Preparation for 3A Ligation and Gels.<br />
<br />
<br />
8/20 Alyssa:<br />
Phosphorylization, annealing, and ligation of mntH, mrgA, spoVG, yvgW, and amyE locus biobricks-- to make biobrick constructs out of the oligos that we ordered<br />
Performed ligation calculations for mrgA promoter, mntH promoter, spoVG, amyE locus, and yvgW promoter<br />
<br />
<br />
<br />
8/30 Malinka: Made Kan plates.<br />
<br />
8/31 Xing: Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/1 Matt: Inoculated mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, and yvgW/spoVG to prepare for a miniprep. The plates he inncoculated from: No L 50ul control looks almost completely clear. No L 250ul control has very small speckling on surface. Both 50ul and 250ul no I controls grew dozens of singles. PUC19 grew. H20 control is clear. All samples grew.<br />
<br />
<br />
9/2 Xing: Miniprep mntH/mrgA rbs, mntH/spoVG, mrgA/mrgArbs, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG<br />
<br />
9/3 Alyssa: Digested mntH/ mrgArbs, mntH/ spoVG rbs, YFP, and GFP<br />
<br />
<br />
9/4 Xing: Ligation of mntH-mrgA/YFP, mntH-spoVG/YFP, mrgAprom-mrgArbs/GFP and transformation<br />
<br />
9/7 Xing: Miniprep cultures of mntH/spoVG/YFP, mntH/mrgArbs/YFP, mrgAprom/spoVG, yvgW/mrgArbs, yvgW/spoVG <br />
<br />
9/8 Malinka: Transforming and plating promoter + RBS bricks (yvgW and mrgAprom with spoVG or mrgArbs).<br />
Matt tested to see if backbones took up desired sequences (mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs) by running a gel. Plasmids cut with E and P show slightly smaller bands than those cut with just E on both gels. Wells 6 and 7 corresponding to mrgApmrgA are very light.<br />
<br />
<br />
9/9 Xing: Send constructs for sequencing<br />
mrgA promoter, mrgA rbs proper sequence. mntH, yvgW, spoVG inserted inverted into plasmid.<br />
<br />
9/11: Miniprep and digestion of yvgW and mrgA constructs<br />
<br />
9/13 Xing: Digestion and Gel Analysis of mntH, yvgW, spoVG parts <br />
spoVG 6:1 2,3 migrate as expected, send for sequencing<br />
Send spoVG 6:1 2,3 for sequencing<br />
Both samples proper sequence<br />
<br />
9/15: Alyssa Miniprepped 9/14 inoculated cultures—yvgW, mntH, spoVG<br />
<br />
<br />
9/16 Xing: Digestion and Gel Analysis of mntH, yvgW<br />
No samples appear linearized<br />
Matt prepared competent DH5alpha E Coli cells. Grew two 50ml cultures to .35 OD600 and .37 OD600.<br />
Pellets re-suspended fine. About 80-85 100ul aliquots of competent cells were stored in -80C.<br />
<br />
9/21 Xing: Ligation of mntH, yvgW inserts into pSB1AC3 backbones<br />
pUC19 control shows colonies, all other plates blank<br />
<br />
9/22 Matt checked to see that the newly transformed plates from 9/21 did not grow, so he inncoulated only from the old 8/13 and 7/22 plates. Taking from colonies that do not appear to have been sampled: 9 colonies from yvgW 6:1 buc 7/22 3 colonies from mntH 6:1 buc 8/13 3 colonies from mntH 6:1 buc 8/13 3 colonies from mntH 6:1 buc 8/13<br />
<br />
<br />
9/24 Malinka: : Digestion (with X) and Gel Analysis of mntH, yvgW parts to determine which promoter biobricks were good and should be sequenced.<br />
Alyssa: Miniprepped mntH, yvgW<br />
<br />
9/25 Malinka: : Preparing mntH, yvgW biobricks for sequencing.<br />
<br />
9/28 Xing: Digestion and Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/29 Malinka: Culturing (mntH and yvgW) + RBS (spoVG) biobricks.<br />
<br />
9/30 Xing: Preparing promoter rbs constructs for sequencing<br />
yvgW/spoVG 1 9/30 displays proper sequence<br />
<br />
10/3 Ben<br />
Digested yvgW/spoVG, CFP, and pSB1AC3 (rfp insert)<br />
Ligated yvgW/spoVG to CFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
<br />
10/6 Matt innoculated from 8/31yvgW/mrgArbs and 10/5 yvgW/spovG/cfp. 10/5 yvgW/spovG/cfp grew on chlor all colonies white many colonies. The chlor controls were clear.<br />
<br />
<br />
10/10 Malinka: Transforming and plating yvgW/spoVG/GFP biobricks and yvgW/spoVG biobricks <br />
Alyssa: <br />
Digested yvgW/spoVG, GFP, PSB1AC3<br />
Ligated promoter/rbs and GFP to backbone<br />
<br />
10/11 Matt streaked 2 plates of B Subtillis Wild Type (CU1065) from frozen permanent stock to grow up for transformation. Nothing grew on either plate, the permanents must be bad. <br />
<br />
10/12 Malinka: 3 way ligation to construct mrgAprom/mrgArbs parts. Re-digesting plasmids to construct yvgW/spoVG/GFP parts<br />
Alyssa: Plated mrgA/ mrgA rbs on Kan plates<br />
<br />
<br />
10/16 Xing: Preparation of Kan, Chlor, LB plates<br />
<br />
<br />
10/17 Xing: Preparing samples for registry<br />
Ben<br />
Digested yvgW/spoVG, GFP, and pSB1AC3 (rfp insert)<br />
Ligated yvgW/spoVG to GFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
Malinka: Preparing samples for registry. Re-transforming and plating yvgW/spoVG/GFP<br />
<br />
10/20: Alyssa :<br />
Miniprepped yvgW/spoVG/GFP on Chlor—pink pellets, so did not miniprep in full to obtain DNA<br />
Inoculated six tubes of colonies plated on 10/17—yvgW/spoVG/GFP on chlor plate.</div>Bammarata89http://2009.igem.org/Team:Cornell/NotebookTeam:Cornell/Notebook2009-10-22T03:37:58Z<p>Bammarata89: </p>
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6/26/09<br />
Alyssa and Matt—made chloramphenicol, kanamycin, and tetracycline antibiotic stock solutions for use in agar plates. See “Protocols” folder for antibiotic making protocol.<br />
<br />
<br />
Antibiotic Volume (mL): Stock (mg/mL): Amnt use (g): Solvent<br />
chloramphenicol 15 34(200x) 0.510 EtOH<br />
kanamycin 15 10(200x) 0.150 H20<br />
tetracycline 50 5(200x) 0.250 EtOH<br />
<br />
Comments: <br />
Please note that tetracycline does NOT fully dissolve in 100% EtOH—will see a yellow powder in a clear solution.<br />
For all stock solutions of antibiotics dissolved in H20: “sterilize by filtration through a 0.22um filter (Sambrook and Russel)”<br />
<br />
Xing—PCR cloning of GFP+ into Biobrick standard A<br />
<br />
6/29/09<br />
<br />
Malinka and Ben—Spread antibiotics on agar plates for ccdb amplification. Created a table for the antibiotics we were going to use and then top spread antibiotics onto agar plates. <br />
<br />
Comments:<br />
The color code for the antibiotics: <br />
Amp – Black<br />
Chlor – Red<br />
Kan – Green<br />
Tet – Blue<br />
Table they came up with is Ben’s notebook entry for the day. Results are summarized as the following: <br />
--pSB1AK3 had colonies on each plate<br />
water plates had no colonies, except for amp plates<br />
pSB1AC3 and pSB1AT3 had no colonies, except for on the amp plates<br />
Xing Agarose Gel Electrophoresis Analysis of GFP+ PCR Product to analyze products of PCR of GFP+<br />
<br />
<br />
Lanes 1 2 3 4 5 6 7 8<br />
PFx 1ng 5λ <br />
PFx 10ng 5λ <br />
PFx 100ng 5λ <br />
Pfu 1ng 5λ <br />
Pfu 10ng 5λ <br />
Pfu 100ng 5λ <br />
pGFP+ 1 5λ <br />
100bp ladder Fermentas 5λ<br />
6X Loading dye 2λ 2λ 2λ 2λ 2λ 2λ 2λ <br />
Total Volume 7λ 7λ 7λ 7λ 7λ 7λ 7λ 5λ<br />
<br />
Comments:<br />
The rxns using Pfx display product at the expected mobility ~750bp with increasing yields following increasing amount of template plasmid used. The rxns treated with Pfu displayed no product.<br />
<br />
6/30/09<br />
<br />
1. Xing—PCR Amplification of yvgY promoter & GFP+. The goal was to PCR Amplify yvgY promoter from chromosome into BioBrick standard and repeat GFP+ amplification since we used 10X more primer in the previous experiment than we should have.<br />
<br />
2. Malinka and Ben—extracted iGEM backbone plasmids from kit (wells 11c for pSB1AC3, 13e for pSB1AK3, and 15c for pSB1AT3) and transformed them. Also grew B. subtilis strains to make frozen permanents. <br />
Comments:<br />
Ben's plates (for pSB1AK3) had various numbers of colonies<br />
Malinka’s plates (for pSB1AC3 and pSB1AT3)<br />
Nothing grew on the 50 ul of only Tet or Chlor<br />
Nothing grew in the 50 ul or 250 ul of the Tet and amp and the Chlor and Amp<br />
No colonies on water plates<br />
Some cells grew on the only amp plates. Thus amp not very effective, and there was a large “background”<br />
<br />
7/1 <br />
Xing; Agarose Gel Electrophoresis of GFP+/yvgY promoter PCR Products<br />
GFP+ bands display expected mobility, with greater yield in the 1ng rxn than the previous rxn. No apparent product in rxns amplified by Pfu. yvgY shows up faintly for rxns using Pfx and Pfu with expected mobility <100bp. <br />
Alyssa and Matt: Growth Curve Characterization of B Subtilis at Different Culture Scales To characterize the growth curves of B Subtilis in both ml scale tubes and in microtiter plates by measuring optical density at set intervals during growth. (By finding when the strains enter logarithmic growth phase, we can better design our B galactosidase assay protocol.) Calculations assume midlog phase.<br />
Ben: Inoculating pSB1AK3 & re-plating pSB1AC3 and pSB1AT3. Results: Inoculating pSB1AK3<br />
All tubes contain cells…SUCCESS! Re-plating pSB1AC3 and pSB1AT3<br />
None of the water plates grew cells, except for the tet lower concentration plate<br />
The supposed plasmid containing cells also did not grow, except for some background on the lower concentration tet plate.<br />
Malinka: Replating plasmids and making frozen permanents Replate the pSB1AC3 and pSB1AT3 and do frozen permanents<br />
Results: Plasmids didn’t grow, lawn on water<br />
<br />
7/2 <br />
Xing: Gel Electrophoresis of yvgY promoter PCR rxn with primers<br />
Compare yvgY PCR rxns with primers to confirm or refute presence of product<br />
Primers do not show up on gel, there is a band below 100bp for the PCR rxns. The expected product size is ~120 bp. The <100 band could be due to primer dimerization. Run a primer titration down to 50nM primer to check next time.<br />
Ben: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain. Results: Re-transforming pSB1AC3 and pSB1AT3-No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates). plating ccdB-resistant strain -2 lawns formed<br />
Malinka: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain<br />
Results: -No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates) plating ccdB-resistant strain-2 lawns formed<br />
<br />
7/3 <br />
Xing: Preparation of Chemically Competent E.Coli. Part II<br />
Xing: Gel Analysis PCR of yvgY and yvgW promoters primer titration; Run agarose gel electrophoresis to analyze PCR rxns yvgY rxns display the same band <100bp but appears less intense for .1ng/uL primer rxn and even less for .05ng/uL primer rxn, supporting the hypothesis that these are primer dimerization products. yvgW does not display any products. Possibility of broken chromosomal DNA? Extract Bacillus DNA next time.<br />
Xing: PCR Amplification of yvgY and yvgW promoter PCR amplify yvgY and yvgW promoter into BB standard and titrate primers to see if proper product forms.<br />
Xing: Prepare Kanamycin and Tetracycline Plates<br />
Ben: Preparation of Chemically Competent E.Coli.<br />
Malinka: Prepare Chloramphenicol and Ampicillin Plates, and grow competent cells<br />
<br />
7/6<br />
Ben and Malinka: Checking competency of cells/ if plates are working (by Malinka). Bad results, redo experiment<br />
7/7<br />
Ben: Re-re-transforming cells (LOL). Transform (hopefully) pSB1AC3 and pSB1AT3 in ccdB-resistant and DH5a strains.<br />
--Results: -Tet plates (including water and both pSB1AT3 plates) contained lawns, reason unkown<br />
-Chlor plates came out as predicted, nothing on water plates, dozens of colonies on 250uL plate, <10 on 50uL plate, but colonies were not red still need to view them under UV light<br />
-water controls were clean, except for Tet, as said above<br />
-pUC19 1:10 dilution had <10 colonies, 1:100 had none visible replating at 1:1 to check competent cells’<br />
Malinka: Replating plasmid backbones-- Playing pSB1AK3 and pSB1AC3 on new antibiotic plates<br />
--Results: pSB1AT3 (ccdb) – Proportional amounts of single colonies had grown on my 50 and 250 ul plates. However a similar amount of colonies had grown on the water too. <br />
<br />
pSB1AT3 (DH5a) – Ben had lawns on all his Tet plates.<br />
<br />
pSB1AC3 (ccdb) – I had nothing on all 3 of my chlor plate.<br />
<br />
pSB1AC3 (DH5a) – Proportional amounts of single colonies had grown on his 50 and 250 ul plates. Nothing had grown on his water. The cells weren’t red like we expected them to be but that was probably because they were too small.<br />
<br />
Water (1:1000) (ccdb and DH5a) – We had lots of single colonies on both our plates. They looked similar.<br />
<br />
pUC19 (ccdb) - <br />
<br />
pUC19 (DH5a) - <br />
<br />
<br />
7/8<br />
Xing: PCR yvgY promoter & yvgW temperature using a temperature gradient<br />
Xing: Preparation of mntR frozen permanents<br />
Ben: Innoculating pSB1AC3 and pSB1AT3 (rfp versions) to see if we have transformed backbones. <br />
--Results: Chlor plasmids had growth, reddish in tube!-Tet did not grow, as expected<br />
Malinka: culture backbone from the plates done yesterday<br />
--Results: All of Ben’s pSB1AC3s grew and turned red. Only 1 of Malinka’s pSB1AT3s grew<br />
Alyssa: Preparing B. subtilis cultures exposed to ZnCl2 for practice LacZ Assay-- To try out the B-Gal assay protocol mentioned on page 12 of Helmann’s “Genetic and physiological responses of B. subtilis to metal ion stress” paper<br />
--Results: Whoa! Heavy metals/ growth retardation in action! According to the last OD600 readings, ZnCl2 seems to significantly (and negatively) affect the growth rates of the B. subtilis strains exposed to the highest concentrations of metal solution.<br />
<br />
7/9 <br />
Xing: Agarose Gel Electrophoresis of PCR yvgY yvgW temperature gradient to analyze PCR products-- yvgY rxn still forms primer dimmers, though concentration seems to decrease with increasing temperature. No products for yvgW rxn.<br />
Matt and Alyssa: Designed Zinc growth curve plate<br />
Ben: Mini-prep pSB1AC3 (rfp) and pSB1AK3 (ccdB)<br />
7/10<br />
Xing: Agarose Gel Electrophoresis of Temperature Gradient to analyze PCR products<br />
Xing: PCR temperature gradient of yvgY & yvgW-- PCR yvgY & yvgW promoters with positive controls.<br />
7/13<br />
Malinka: Digesting the plasmid backbones PSB1AC3 and PSB1AK3<br />
<br />
7/14<br />
Xing: Gel electrophoresis of varying [Mg2+] PCR<br />
Xing: PCR Amplification of yvgY & yvgW w/ varying [Mg2+]-- Vary [Mg2+] in PCR rxn to see if products form<br />
Malinka: ran gels for backbones<br />
7/15<br />
Matt: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
Malinka: Digestion - Removing inserts from the plasmid backbones PSB1AC3, PSB1AK3, PSB1AT3<br />
--Results: <br />
<br />
7/16<br />
Ben: DNA electrophoresis of doubly digested (X & S) backbones and GFP+<br />
7/17<br />
Xing: PNK treatment and annealing of complementary primers-- PNK treat primers to phosphorylate ends for ligation/anneal complementary primers<br />
Alyssa: CdCl2 soln growth curve plate design for use in TECAN machine<br />
7/20<br />
Ben: Check of pSB1AK3 ccdB-gene effectiveness through transformation in DH5a<br />
--Results: -waters were blank-puc19 had a few cells -sSB1AK3 plates both transformed, meaning the ccdB gene is broken, since the cells should have died<br />
Malinka: Dephosphoralating and purifying backbone plasmids PSB1AC3, PSB1AK3, PSB1AT3<br />
7/21<br />
Matt and Alyssa: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
Ben: Ligation of promoters and GFP+<br />
Malinka: Creating GFP and promoter biobricks—yvgW and yvgY<br />
--Results: Based on yesterdays Tet plate results, we found that the Tet plates work. But the ccdb psb1AK3 does not work so we have only pSB1AC3 and pSB1AT3 as backbones.<br />
7/22<br />
Ben: Transformation of ligations yvgW and GFP+<br />
Malinka: Redigestion - Removing inserts from the plasmid backbones<br />
Results: Plated Ligated Biobricks <br />
<br />
Controls:<br />
The pUC19 on amp showed reasonable growth and the water on amp had nothing which meant that there was no background on the amp plates. As we still got good growth for pUC19, this showed that DH5 cells were alive and competent. The water on the Chlor plater had no growth. <br />
<br />
The no insert and no enzyme ligation mixtures showed no growth.<br />
<br />
yvgW and yvgY:<br />
There was little growth on the 2:1 ratio plates for yvgW and yvgY (maybe 5 colonies). There was more growth on the 6:1 ratio plates (maybe 15 colonies) – we plan to use 6:1 ratio from now on.<br />
<br />
GFP:<br />
There was no growth on any of the GFP biobrick plates (2:1, 4:1 or 6:1).<br />
<br />
Undigested plasmid (pSB1AC3) would have shown up as red cells on the plate. However from previous experience, we knew that 24 hours of incubation wasn't enough for the cells to have grown large enough or long enough to be visibly red. So we weren't sure, of the plates with growth in them, which cells actually had been transformed with our biobricks as opposed to the undigested plasmid. <br />
<br />
7/23<br />
Ben: Transformation of pSB1K3 and pSB1T3 backbones<br />
--Results: -Kan plates produced colonies (slightly red) -waters had nothing (except tet plates bad plates) -pUC19 had colonies -tet plates all had lawns bad plates<br />
Malinka: To run gels on the newly digested plasmid backbones and cut GFP to make sure we had got them right. To dephosphorylate the cut backbones and then purify them so that we had enough vector to ligate with our inserts later.<br />
--Results: The gels looked good for Chlor, Tet and Kan. I was even able to see a the insert for Kan very low in the gel (it was very faint) – exactly where the iGEM gel showed it to be.<br />
<br />
I got nothing for the cut and uncut GFP. Ben had used uncut GFP from the same tube a few days ago and got results but he used 1 ul which is (600-700 ng) so perhaps I had too little. We need to do this again with more cut and uncut GFP.<br />
<br />
<br />
Xing – To culture tubes with colonies from our transformed biobricks (with promoters). Also to culture 1 tube with pSB1AC3 (with RFP) because we were going to use RFP (and GFP) for our project. <br />
<br />
Ben and Matt – To make new Chlor and Tet plates and transform cells with new backbone plasmid from the kit (pSB1T3 & pSB1K3) in case our next backbone doesn't work. <br />
7/24<br />
Cadmium and Zinc Beta Galactosidase Assay in tubes: To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion. Couldn’t access spectrometer when the assay was complete, so tubes were stopped when they showed a clear yellow color and left to sit out over the weekend. On Monday all remaining tubes were stopped and OD420, OD550, and OD600 were measured in the spec.<br />
Malinka: Test--Digesting the pSB1AC3 and pSB1AT3 using 15 minute method and digesting purified GFP+<br />
--Results: The 15 minute digest of pSB1AC3 and pSB1AT3 showed the same results as the digests of the same plasmids done overnight.<br />
<br />
We saw the uncut GFP but did not see the cut GFP. We think it’s because the GFP+ wasn’t purified.<br />
<br />
<br />
<br />
7/24<br />
Xing: Purification of GFP+ PCR Products<br />
<br />
7/27<br />
Xing; Purification of Additional GFP+ PCR Products<br />
Alyssa and Malinka: Digesting GFP+ and running gels on it-- To test pure cut GFP+ digested for 6 hours, cut and then test GFP+ digested for 15 minutes, cut and then test new backbones, and cut and then test biobrick parts with promoters.<br />
7/29<br />
Ben: Mini-prep of GFP (E0040), CFP (E0020), and YFP (E0030)<br />
Alyssa: designed primers to extract constitutive mrgA promoter from Helmann strain of B. sub.<br />
7/30<br />
Alyssa and Xing: Digest pSB1K3 & ligate GFP+ part onto digest--Digestion of assembly vector pSB1K3, dephosphorylation & ligation of GFP+ onto assembly vector. <br />
Ben: Digestion of GFP CFP and YFP-- Appropriate bands located in all wells: perfect!<br />
<br />
<br />
<br />
7/31<br />
Xing: Transformation of GFP+ Ligation and selection with Kanamycin<br />
Xing: Transformation of pSB1AC3 using Amp selection--1 Colony for 50uL pSB1AC3<br />
~ 10 Colonies for 250 uL pSB1AC3, no colonies for H2O, pUC19 250 uL 0-5 colonies.<br />
Alyssa and Ben: Ligation of assembly vector PSBK3 to GFP+ to create GFP+ biobrick parts<br />
<br />
8/3<br />
Xing: Digest & gel analysis of yvgW, yvgY, GFP+ BB parts<br />
- No GFP+ parts, incomplete digestion for yvgW, yvgY parts.<br />
Malinka: Mini-prepped GFP+ biobricks and pSB1AC3<br />
8/4 Xing: Digest & gel analysis of yvgW, yvgY BB parts<br />
- Mobility as expected for for yvgW 2:1 1 and yvgY 6:1 3<br />
Malinka: Transformed and plated pSB1AT3 (rfp and ccdb) from the kit.<br />
<br />
8/5 Xing: Testing effectiveness of tetracycline plates<br />
- Lawn formed on all tet plates<br />
Malinka: Cultured pSB1AT3 (DH5) cells, and yvgY (on pSB1AC3), yvgW (on pSB1AC3) and GFP+ (on pSB1AK3) biobricks.<br />
Matt: Previous Tetracyclin plates have failed to kill strains without Tet resistance. Matt made another attempt to create working Tetracyclin using a new improved stock of 100X Tet. A new Tetracycline stock was created. Previous stocks were made using 100% Ethanol under the assumption that we were working with pure Tetracyline. However, our solid stock is actually Tetracyline Hydrochloride. This compound has difficulty suspending in pure Ethanol. Using a 70% Ethanol 30% water solvent the Tetracyline Hydrochloride dissolved almost instantly. Previously no precautions were made to counter Tetracyline’s light sensitivity. This time we wrapped the stock in tin foil and were careful to avoid too much light exposure.<br />
<br />
<br />
Digest & gel analysis of yvgW 2:1 1, yvgY 6:1 3, GFP+ 2:1 50 BB parts<br />
- yvgW 2:1 1 sample with expected mobility, yvgY 6:1 3 sample incompletely digested, GFP+ 2:1 50 sample not as expected<br />
<br />
8/6 Matt characterize Tet’s effect on cells when top spreading and in liquid culture. All of the 0x, .1x, and 1x Tet tubes grew to saturation while neither of the 10x Tet tubes grew. Further testing is necessary to determine if a concentration can be found where Tet resistant strains grow but strains without Tet resistance do not grow. All of the plates grew. However, 1X Tet with Tet resistant strain PSB1AT3 in DH5alpha did not grow to a lawn but showed many (>100) small single colonies.<br />
<br />
8/7 Matt: Previously we determined that both Tet resistant and unresistant cells grow in 1x Tet liquid culture but die in 10x liquid culture. Our purpose is to determine a Tet concentration between these two extremes where Tet resistant culture lives and Tet unresistant culture does not. Cultures were cloudy for pSB1AT3 & pUC19: 0X tet, 2X tet. Cultures were clear for pSB1AT3 & pUC 19: 4X tet, 6X tet, 8X tet. OD600 for pSB1AT3 2X tet(10X dilution): 0.24 OD600 for pUC19 2X tet(10X dilution): 0.18 Not a significant OD600 difference to select from. Test range from 2X-4X tet next.<br />
<br />
<br />
<br />
8/10 Xing: Transformation of BB_spoVG RBS & BB_mrgApromoterrbs<br />
Failed transformation<br />
Alyssa: Made Excel template for ligation calculations. Phosphorylated, annealed, and ligated purchased oligonucleotides:<br />
mrgA, mntH, spoVG1, spoVG2<br />
<br />
Malinka: Phosphorylated and annealed mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS<br />
Matt: Previously we determined that both Tet resistant and unresistant cells grow in 1x Tet liquid culture but die in 10x liquid culture. Our purpose is to determine a Tet concentration between these two extremes where Tet resistant culture lives and Tet unresistant culture does not. Using strains: PSB1AT3 in DH5alpha and PUC19 in DH5alpha at concentration of 0X, 2.5X, 3X, 3.5x, and 4X Tet<br />
(1X Tet= 50ug/ml, stock is at 100X) Culture in 5ml of LB, inoculate from picking plates. At 2.5X PSB1At3 DH5alpha grew but PUC19 DH5alpha did not. Above this concentration nothing grew.<br />
<br />
<br />
<br />
8/11 Malinka: : Transformed and plated mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS (all on pSB1AC3).<br />
Alyssa: Ligated and transformed oligos of mntH promoter and mrgA promoter. Transformed and plated RBS and mnth promoter biobricks in DH5 cells<br />
<br />
<br />
<br />
8/12 Xing: Test transformation protocol and competency of cells <br />
Cells competent and transformation worked with pUC19<br />
Alyssa: CdCl2 growth curve plate design<br />
<br />
<br />
8/13 Xing: Tranformation of mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
Successful transformation<br />
Ben: Inoculated spoVG transformants for mini-prepping<br />
Malinka: Cultured amyE parts and made amp plates.<br />
Alyssa:<br />
Redid CdCl2 growth curves redo the CdCl2 growth curve experiment from 7/18, which resulted in biphasic growth curves and growth of mntR when exposed to Cd. (This should not have happened due to its extreme Cd sensitivity.)<br />
--Results:<br />
No errors <br />
111 runs<br />
Biphasic growth curves for all strains, though not as pronounced as 1st growth curve exp.<br />
mntR shows more growth than WT, even at 5uM Cd <br />
<br />
<br />
<br />
8/14 Xing: Inoculate cultures from transformant plates for mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
PAGE purification of mrgApromrbs<br />
Alyssa:<br />
Made new 1000X Tet stock and measured concentration of PSB1K3-- to make a more potent Tet stock soln. and measure concentration of a biobrick backbone for calculation purposes in other experiments and procedures (such as ligation).<br />
Analyzed data from 8/13 growth curves on Excel<br />
Cut and dephosphorylated psB1K3 backbone<br />
<br />
<br />
8/15 Xing: Digest and gel analysis of spoVG, BBa_K143001, BBa_K143002<br />
spoVG migrates with expected mobility, BBa_K143001 & 2 do not migrate as expected<br />
<br />
Miniprep mntH, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
8/19 Alyssa: Digestion of mntH, mnrA promoter, mrgA + mrgA rbs, and mrgA rbs<br />
Matt: digested Biobricks in Preparation for 3A Ligation and Gels.<br />
<br />
<br />
8/20 Alyssa:<br />
Phosphorylization, annealing, and ligation of mntH, mrgA, spoVG, yvgW, and amyE locus biobricks-- to make biobrick constructs out of the oligos that we ordered<br />
Performed ligation calculations for mrgA promoter, mntH promoter, spoVG, amyE locus, and yvgW promoter<br />
<br />
<br />
<br />
8/30 Malinka: Made Kan plates.<br />
<br />
8/31 Xing: Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/1 Matt: Inoculated mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, and yvgW/spoVG to prepare for a miniprep. The plates he inncoculated from: No L 50ul control looks almost completely clear. No L 250ul control has very small speckling on surface. Both 50ul and 250ul no I controls grew dozens of singles. PUC19 grew. H20 control is clear. All samples grew.<br />
<br />
<br />
9/2 Xing: Miniprep mntH/mrgA rbs, mntH/spoVG, mrgA/mrgArbs, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG<br />
<br />
9/3 Alyssa: Digested mntH/ mrgArbs, mntH/ spoVG rbs, YFP, and GFP<br />
<br />
<br />
9/4 Xing: Ligation of mntH-mrgA/YFP, mntH-spoVG/YFP, mrgAprom-mrgArbs/GFP and transformation<br />
<br />
9/7 Xing: Miniprep cultures of mntH/spoVG/YFP, mntH/mrgArbs/YFP, mrgAprom/spoVG, yvgW/mrgArbs, yvgW/spoVG <br />
<br />
9/8 Malinka: Transforming and plating promoter + RBS bricks (yvgW and mrgAprom with spoVG or mrgArbs).<br />
Matt tested to see if backbones took up desired sequences (mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs) by running a gel. Plasmids cut with E and P show slightly smaller bands than those cut with just E on both gels. Wells 6 and 7 corresponding to mrgApmrgA are very light.<br />
<br />
<br />
9/9 Xing: Send constructs for sequencing<br />
mrgA promoter, mrgA rbs proper sequence. mntH, yvgW, spoVG inserted inverted into plasmid.<br />
<br />
9/11: Miniprep and digestion of yvgW and mrgA constructs<br />
<br />
9/13 Xing: Digestion and Gel Analysis of mntH, yvgW, spoVG parts <br />
spoVG 6:1 2,3 migrate as expected, send for sequencing<br />
Send spoVG 6:1 2,3 for sequencing<br />
Both samples proper sequence<br />
<br />
9/15: Alyssa Miniprepped 9/14 inoculated cultures—yvgW, mntH, spoVG<br />
<br />
<br />
9/16 Xing: Digestion and Gel Analysis of mntH, yvgW<br />
No samples appear linearized<br />
Matt prepared competent DH5alpha E Coli cells. Grew two 50ml cultures to .35 OD600 and .37 OD600.<br />
Pellets re-suspended fine. About 80-85 100ul aliquots of competent cells were stored in -80C.<br />
<br />
9/21 Xing: Ligation of mntH, yvgW inserts into pSB1AC3 backbones<br />
pUC19 control shows colonies, all other plates blank<br />
<br />
9/22 Matt checked to see that the newly transformed plates from 9/21 did not grow, so he inncoulated only from the old 8/13 and 7/22 plates. Taking from colonies that do not appear to have been sampled: 9 colonies from yvgW 6:1 buc 7/22 3 colonies from mntH 6:1 buc 8/13 3 colonies from mntH 6:1 buc 8/13 3 colonies from mntH 6:1 buc 8/13<br />
<br />
<br />
9/24 Malinka: : Digestion (with X) and Gel Analysis of mntH, yvgW parts to determine which promoter biobricks were good and should be sequenced.<br />
Alyssa: Miniprepped mntH, yvgW<br />
<br />
9/25 Malinka: : Preparing mntH, yvgW biobricks for sequencing.<br />
<br />
9/28 Xing: Digestion and Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/29 Malinka: Culturing (mntH and yvgW) + RBS (spoVG) biobricks.<br />
<br />
9/30 Xing: Preparing promoter rbs constructs for sequencing<br />
yvgW/spoVG 1 9/30 displays proper sequence<br />
<br />
10/3 Ben<br />
Digested yvgW/spoVG, CFP, and pSB1AC3 (rfp insert)<br />
Ligated yvgW/spoVG to CFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
<br />
10/6 Matt innoculated from 8/31yvgW/mrgArbs and 10/5 yvgW/spovG/cfp. 10/5 yvgW/spovG/cfp grew on chlor all colonies white many colonies. The chlor controls were clear.<br />
<br />
<br />
10/10 Malinka: Transforming and plating yvgW/spoVG/GFP biobricks and yvgW/spoVG biobricks <br />
Alyssa: <br />
Digested yvgW/spoVG, GFP, PSB1AC3<br />
Ligated promoter/rbs and GFP to backbone<br />
<br />
10/11 Matt streaked 2 plates of B Subtillis Wild Type (CU1065) from frozen permanent stock to grow up for transformation. Nothing grew on either plate, the permanents must be bad. <br />
<br />
10/12 Malinka: 3 way ligation to construct mrgAprom/mrgArbs parts. Re-digesting plasmids to construct yvgW/spoVG/GFP parts<br />
Alyssa: Plated mrgA/ mrgA rbs on Kan plates<br />
<br />
<br />
10/16 Xing: Preparation of Kan, Chlor, LB plates<br />
<br />
<br />
10/17 Xing: Preparing samples for registry<br />
Ben<br />
Digested yvgW/spoVG, GFP, and pSB1AC3 (rfp insert)<br />
Ligated yvgW/spoVG to GFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
Malinka: Preparing samples for registry. Re-transforming and plating yvgW/spoVG/GFP<br />
<br />
10/20: Alyssa :<br />
Miniprepped yvgW/spoVG/GFP on Chlor—pink pellets, so did not miniprep in full to obtain DNA<br />
Inoculated six tubes of colonies plated on 10/17—yvgW/spoVG/GFP on chlor plate.</div>Bammarata89http://2009.igem.org/Team:Cornell/NotebookTeam:Cornell/Notebook2009-10-22T03:35:02Z<p>Bammarata89: </p>
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6/26/09<br />
Alyssa and Matt—made chloramphenicol, kanamycin, and tetracycline antibiotic stock solutions for use in agar plates. See “Protocols” folder for antibiotic-making protocol.<br />
<br />
<br />
Antibiotic Volume (mL): Stock (mg/mL): Amnt use (g): Solvent<br />
chloramphenicol 15 34(200x) 0.510 EtOH<br />
kanamycin 15 10(200x) 0.150 H20<br />
tetracycline 50 5(200x) 0.250 EtOH<br />
<br />
Comments: <br />
Please note that tetracycline does NOT fully dissolve in 100% EtOH—will see a yellow powder in a clear solution.<br />
For all stock solutions of antibiotics dissolved in H20: “sterilize by filtration through a 0.22um filter (Sambrook and Russel)”<br />
<br />
Xing—PCR cloning of GFP+ into Biobrick standard A<br />
<br />
6/29/09<br />
<br />
Malinka and Ben—Spread antibiotics on agar plates for ccdb amplification. Created a table for the antibiotics we were going to use and then top-spread antibiotics onto agar plates. <br />
<br />
Comments:<br />
The color code for the antibiotics: <br />
Amp – Black<br />
Chlor – Red<br />
Kan – Green<br />
Tet – Blue<br />
Table they came up with is Ben’s notebook entry for the day. Results are summarized as the following: <br />
--pSB1AK3 had colonies on each plate<br />
--water plates had no colonies, except for amp plates<br />
--pSB1AC3 and pSB1AT3 had no colonies, except for on the amp plates<br />
Xing-- Agarose Gel Electrophoresis Analysis of GFP+ PCR Product to analyze products of PCR of GFP+<br />
<br />
<br />
Lanes 1 2 3 4 5 6 7 8<br />
PFx 1ng 5λ <br />
PFx 10ng 5λ <br />
PFx 100ng 5λ <br />
Pfu 1ng 5λ <br />
Pfu 10ng 5λ <br />
Pfu 100ng 5λ <br />
pGFP+ 1 5λ <br />
100bp ladder Fermentas 5λ<br />
6X Loading dye 2λ 2λ 2λ 2λ 2λ 2λ 2λ <br />
Total Volume 7λ 7λ 7λ 7λ 7λ 7λ 7λ 5λ<br />
<br />
Comments:<br />
The rxns using Pfx display product at the expected mobility ~750bp with increasing yields following increasing amount of template plasmid used. The rxns treated with Pfu displayed no product.<br />
<br />
6/30/09<br />
<br />
1. Xing—PCR Amplification of yvgY promoter & GFP+. The goal was to PCR Amplify yvgY promoter from chromosome into BioBrick standard and repeat GFP+ amplification since we used 10X more primer in the previous experiment than we should have.<br />
<br />
2. Malinka and Ben—extracted iGEM backbone plasmids from kit (wells 11c for pSB1AC3, 13e for pSB1AK3, and 15c for pSB1AT3) and transformed them. Also grew B. subtilis strains to make frozen permanents. <br />
Comments:<br />
Ben's plates (for pSB1AK3) had various numbers of colonies<br />
Malinka’s plates (for pSB1AC3 and pSB1AT3)<br />
--Nothing grew on the 50 ul of only Tet or Chlor<br />
--Nothing grew in the 50 ul or 250 ul of the Tet and amp and the Chlor and <br />
Amp<br />
--No colonies on water plates<br />
--Some cells grew on the only amp plates. Thus amp not very effective, and <br />
there was a large “background”<br />
<br />
7/1 <br />
Xing; Agarose Gel Electrophoresis of GFP+/yvgY promoter PCR Products<br />
GFP+ bands display expected mobility, with greater yield in the 1ng rxn than the previous rxn. No apparent product in rxns amplified by Pfu. yvgY shows up faintly for rxns using Pfx and Pfu with expected mobility <100bp. <br />
Alyssa and Matt: Growth Curve Characterization of B Subtilis at Different Culture Scales--To characterize the growth curves of B Subtilis in both ml scale tubes and in microtiter plates by measuring optical density at set intervals during growth. (By finding when the strains enter logarithmic growth phase, we can better design our B-galactosidase assay protocol.) Calculations assume midlog phase.<br />
Ben: Inoculating pSB1AK3 & re-plating pSB1AC3 and pSB1AT3. Results: Inoculating pSB1AK3<br />
-All tubes contain cells…SUCCESS! Re-plating pSB1AC3 and pSB1AT3<br />
-None of the water plates grew cells, except for the tet lower concentration plate<br />
-The supposed plasmid containing cells also did not grow, except for some background on the lower concentration tet plate.<br />
Malinka: Replating plasmids and making frozen permanents-- Replate the pSB1AC3 and pSB1AT3 and do frozen permanents<br />
--Results: Plasmids didn’t grow, lawn on water<br />
<br />
7/2 <br />
Xing: Gel Electrophoresis of yvgY promoter PCR rxn with primers<br />
Compare yvgY PCR rxns with primers to confirm or refute presence of product<br />
Primers do not show up on gel, there is a band below 100bp for the PCR rxns. The expected product size is ~120 bp. The <100 band could be due to primer dimerization. Run a primer titration down to 50nM primer to check next time.<br />
Ben: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain. Results: Re-transforming pSB1AC3 and pSB1AT3-No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates). plating ccdB-resistant strain -2 lawns formed<br />
Malinka: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain<br />
--Results: -No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates) plating ccdB-resistant strain-2 lawns formed<br />
<br />
7/3 <br />
Xing: Preparation of Chemically Competent E.Coli. Part II<br />
Xing: Gel Analysis PCR of yvgY and yvgW promoters primer titration; Run agarose gel electrophoresis to analyze PCR rxns-- yvgY rxns display the same band <100bp but appears less intense for .1ng/uL primer rxn and even less for .05ng/uL primer rxn, supporting the hypothesis that these are primer dimerization products. yvgW does not display any products. Possibility of broken chromosomal DNA? Extract Bacillus DNA next time.<br />
Xing: PCR Amplification of yvgY and yvgW promoter-- PCR amplify yvgY and yvgW promoter into BB standard and titrate primers to see if proper product forms.<br />
Xing: Prepare Kanamycin and Tetracycline Plates<br />
Ben: Preparation of Chemically Competent E.Coli.<br />
Malinka: Prepare Chloramphenicol and Ampicillin Plates, and grow competent cells<br />
<br />
7/6<br />
Ben and Malinka: Checking competency of cells/ if plates are working (by Malinka). Bad results, redo experiment<br />
7/7<br />
Ben: Re-re-transforming cells (LOL). Transform (hopefully) pSB1AC3 and pSB1AT3 in ccdB-resistant and DH5a strains.<br />
--Results: -Tet plates (including water and both pSB1AT3 plates) contained lawns, reason unkown<br />
-Chlor plates came out as predicted, nothing on water plates, dozens of colonies on 250uL plate, <10 on 50uL plate, but colonies were not red still need to view them under UV light<br />
-water controls were clean, except for Tet, as said above<br />
-pUC19 1:10 dilution had <10 colonies, 1:100 had none visible replating at 1:1 to check competent cells’<br />
Malinka: Replating plasmid backbones-- Playing pSB1AK3 and pSB1AC3 on new antibiotic plates<br />
--Results: pSB1AT3 (ccdb) – Proportional amounts of single colonies had grown on my 50 and 250 ul plates. However a similar amount of colonies had grown on the water too. <br />
<br />
pSB1AT3 (DH5a) – Ben had lawns on all his Tet plates.<br />
<br />
pSB1AC3 (ccdb) – I had nothing on all 3 of my chlor plate.<br />
<br />
pSB1AC3 (DH5a) – Proportional amounts of single colonies had grown on his 50 and 250 ul plates. Nothing had grown on his water. The cells weren’t red like we expected them to be but that was probably because they were too small.<br />
<br />
Water (1:1000) (ccdb and DH5a) – We had lots of single colonies on both our plates. They looked similar.<br />
<br />
pUC19 (ccdb) - <br />
<br />
pUC19 (DH5a) - <br />
<br />
<br />
7/8<br />
Xing: PCR yvgY promoter & yvgW temperature using a temperature gradient<br />
Xing: Preparation of mntR frozen permanents<br />
Ben: Innoculating pSB1AC3 and pSB1AT3 (rfp versions) to see if we have transformed backbones. <br />
--Results: Chlor plasmids had growth, reddish in tube!-Tet did not grow, as expected<br />
Malinka: culture backbone from the plates done yesterday<br />
--Results: All of Ben’s pSB1AC3s grew and turned red. Only 1 of Malinka’s pSB1AT3s grew<br />
Alyssa: Preparing B. subtilis cultures exposed to ZnCl2 for practice LacZ Assay-- To try out the B-Gal assay protocol mentioned on page 12 of Helmann’s “Genetic and physiological responses of B. subtilis to metal ion stress” paper<br />
--Results: Whoa! Heavy metals/ growth retardation in action! According to the last OD600 readings, ZnCl2 seems to significantly (and negatively) affect the growth rates of the B. subtilis strains exposed to the highest concentrations of metal solution.<br />
<br />
7/9 <br />
Xing: Agarose Gel Electrophoresis of PCR yvgY yvgW temperature gradient to analyze PCR products-- yvgY rxn still forms primer dimmers, though concentration seems to decrease with increasing temperature. No products for yvgW rxn.<br />
Matt and Alyssa: Designed Zinc growth curve plate<br />
Ben: Mini-prep pSB1AC3 (rfp) and pSB1AK3 (ccdB)<br />
7/10<br />
Xing: Agarose Gel Electrophoresis of Temperature Gradient to analyze PCR products<br />
Xing: PCR temperature gradient of yvgY & yvgW-- PCR yvgY & yvgW promoters with positive controls.<br />
7/13<br />
Malinka: Digesting the plasmid backbones PSB1AC3 and PSB1AK3<br />
<br />
7/14<br />
Xing: Gel electrophoresis of varying [Mg2+] PCR<br />
Xing: PCR Amplification of yvgY & yvgW w/ varying [Mg2+]-- Vary [Mg2+] in PCR rxn to see if products form<br />
Malinka: ran gels for backbones<br />
7/15<br />
Matt: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
Malinka: Digestion - Removing inserts from the plasmid backbones PSB1AC3, PSB1AK3, PSB1AT3<br />
--Results: <br />
<br />
7/16<br />
Ben: DNA electrophoresis of doubly digested (X & S) backbones and GFP+<br />
7/17<br />
Xing: PNK treatment and annealing of complementary primers-- PNK treat primers to phosphorylate ends for ligation/anneal complementary primers<br />
Alyssa: CdCl2 soln growth curve plate design for use in TECAN machine<br />
7/20<br />
Ben: Check of pSB1AK3 ccdB-gene effectiveness through transformation in DH5a<br />
--Results: -waters were blank-puc19 had a few cells -sSB1AK3 plates both transformed, meaning the ccdB gene is broken, since the cells should have died<br />
Malinka: Dephosphoralating and purifying backbone plasmids PSB1AC3, PSB1AK3, PSB1AT3<br />
7/21<br />
Matt and Alyssa: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
Ben: Ligation of promoters and GFP+<br />
Malinka: Creating GFP and promoter biobricks—yvgW and yvgY<br />
--Results: Based on yesterdays Tet plate results, we found that the Tet plates work. But the ccdb psb1AK3 does not work so we have only pSB1AC3 and pSB1AT3 as backbones.<br />
7/22<br />
Ben: Transformation of ligations yvgW and GFP+<br />
Malinka: Redigestion - Removing inserts from the plasmid backbones<br />
Results: Plated Ligated Biobricks <br />
<br />
Controls:<br />
The pUC19 on amp showed reasonable growth and the water on amp had nothing which meant that there was no background on the amp plates. As we still got good growth for pUC19, this showed that DH5 cells were alive and competent. The water on the Chlor plater had no growth. <br />
<br />
The no insert and no enzyme ligation mixtures showed no growth.<br />
<br />
yvgW and yvgY:<br />
There was little growth on the 2:1 ratio plates for yvgW and yvgY (maybe 5 colonies). There was more growth on the 6:1 ratio plates (maybe 15 colonies) – we plan to use 6:1 ratio from now on.<br />
<br />
GFP:<br />
There was no growth on any of the GFP biobrick plates (2:1, 4:1 or 6:1).<br />
<br />
Undigested plasmid (pSB1AC3) would have shown up as red cells on the plate. However from previous experience, we knew that 24 hours of incubation wasn't enough for the cells to have grown large enough or long enough to be visibly red. So we weren't sure, of the plates with growth in them, which cells actually had been transformed with our biobricks as opposed to the undigested plasmid. <br />
<br />
7/23<br />
Ben: Transformation of pSB1K3 and pSB1T3 backbones<br />
--Results: -Kan plates produced colonies (slightly red) -waters had nothing (except tet plates bad plates) -pUC19 had colonies -tet plates all had lawns bad plates<br />
Malinka: To run gels on the newly digested plasmid backbones and cut GFP to make sure we had got them right. To dephosphorylate the cut backbones and then purify them so that we had enough vector to ligate with our inserts later.<br />
--Results: The gels looked good for Chlor, Tet and Kan. I was even able to see a the insert for Kan very low in the gel (it was very faint) – exactly where the iGEM gel showed it to be.<br />
<br />
I got nothing for the cut and uncut GFP. Ben had used uncut GFP from the same tube a few days ago and got results but he used 1 ul which is (600-700 ng) so perhaps I had too little. We need to do this again with more cut and uncut GFP.<br />
<br />
<br />
Xing – To culture tubes with colonies from our transformed biobricks (with promoters). Also to culture 1 tube with pSB1AC3 (with RFP) because we were going to use RFP (and GFP) for our project. <br />
<br />
Ben and Matt – To make new Chlor and Tet plates and transform cells with new backbone plasmid from the kit (pSB1T3 & pSB1K3) in case our next backbone doesn't work. <br />
7/24<br />
Cadmium and Zinc Beta Galactosidase Assay in tubes: To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion. Couldn’t access spectrometer when the assay was complete, so tubes were stopped when they showed a clear yellow color and left to sit out over the weekend. On Monday all remaining tubes were stopped and OD420, OD550, and OD600 were measured in the spec.<br />
Malinka: Test--Digesting the pSB1AC3 and pSB1AT3 using 15 minute method and digesting purified GFP+<br />
--Results: The 15 minute digest of pSB1AC3 and pSB1AT3 showed the same results as the digests of the same plasmids done overnight.<br />
<br />
We saw the uncut GFP but did not see the cut GFP. We think it’s because the GFP+ wasn’t purified.<br />
<br />
<br />
<br />
7/24<br />
Xing: Purification of GFP+ PCR Products<br />
<br />
7/27<br />
Xing; Purification of Additional GFP+ PCR Products<br />
Alyssa and Malinka: Digesting GFP+ and running gels on it-- To test pure cut GFP+ digested for 6 hours, cut and then test GFP+ digested for 15 minutes, cut and then test new backbones, and cut and then test biobrick parts with promoters.<br />
7/29<br />
Ben: Mini-prep of GFP (E0040), CFP (E0020), and YFP (E0030)<br />
Alyssa: designed primers to extract constitutive mrgA promoter from Helmann strain of B. sub.<br />
7/30<br />
Alyssa and Xing: Digest pSB1K3 & ligate GFP+ part onto digest--Digestion of assembly vector pSB1K3, dephosphorylation & ligation of GFP+ onto assembly vector. <br />
Ben: Digestion of GFP CFP and YFP-- Appropriate bands located in all wells: perfect!<br />
<br />
<br />
<br />
7/31<br />
Xing: Transformation of GFP+ Ligation and selection with Kanamycin<br />
Xing: Transformation of pSB1AC3 using Amp selection--1 Colony for 50uL pSB1AC3<br />
~ 10 Colonies for 250 uL pSB1AC3, no colonies for H2O, pUC19 250 uL 0-5 colonies.<br />
Alyssa and Ben: Ligation of assembly vector PSBK3 to GFP+ to create GFP+ biobrick parts<br />
<br />
8/3<br />
Xing: Digest & gel analysis of yvgW, yvgY, GFP+ BB parts<br />
- No GFP+ parts, incomplete digestion for yvgW, yvgY parts.<br />
Malinka: Mini-prepped GFP+ biobricks and pSB1AC3<br />
8/4 Xing: Digest & gel analysis of yvgW, yvgY BB parts<br />
- Mobility as expected for for yvgW 2:1 1 and yvgY 6:1 3<br />
Malinka: Transformed and plated pSB1AT3 (rfp and ccdb) from the kit.<br />
<br />
8/5 Xing: Testing effectiveness of tetracycline plates<br />
- Lawn formed on all tet plates<br />
Malinka: Cultured pSB1AT3 (DH5) cells, and yvgY (on pSB1AC3), yvgW (on pSB1AC3) and GFP+ (on pSB1AK3) biobricks.<br />
Matt: Previous Tetracyclin plates have failed to kill strains without Tet resistance. Matt made another attempt to create working Tetracyclin using a new improved stock of 100X Tet. A new Tetracycline stock was created. Previous stocks were made using 100% Ethanol under the assumption that we were working with pure Tetracyline. However, our solid stock is actually Tetracyline Hydrochloride. This compound has difficulty suspending in pure Ethanol. Using a 70% Ethanol 30% water solvent the Tetracyline Hydrochloride dissolved almost instantly. Previously no precautions were made to counter Tetracyline’s light sensitivity. This time we wrapped the stock in tin foil and were careful to avoid too much light exposure.<br />
<br />
<br />
Digest & gel analysis of yvgW 2:1 1, yvgY 6:1 3, GFP+ 2:1 50 BB parts<br />
- yvgW 2:1 1 sample with expected mobility, yvgY 6:1 3 sample incompletely digested, GFP+ 2:1 50 sample not as expected<br />
<br />
8/6 Matt characterize Tet’s effect on cells when top spreading and in liquid culture. All of the 0x, .1x, and 1x Tet tubes grew to saturation while neither of the 10x Tet tubes grew. Further testing is necessary to determine if a concentration can be found where Tet resistant strains grow but strains without Tet resistance do not grow. All of the plates grew. However, 1X Tet with Tet resistant strain PSB1AT3 in DH5alpha did not grow to a lawn but showed many (>100) small single colonies.<br />
<br />
8/7 Matt: Previously we determined that both Tet resistant and unresistant cells grow in 1x Tet liquid culture but die in 10x liquid culture. Our purpose is to determine a Tet concentration between these two extremes where Tet resistant culture lives and Tet unresistant culture does not. Cultures were cloudy for pSB1AT3 & pUC19: 0X tet, 2X tet. Cultures were clear for pSB1AT3 & pUC 19: 4X tet, 6X tet, 8X tet. OD600 for pSB1AT3 2X tet(10X dilution): 0.24 OD600 for pUC19 2X tet(10X dilution): 0.18 Not a significant OD600 difference to select from. Test range from 2X-4X tet next.<br />
<br />
<br />
<br />
8/10 Xing: Transformation of BB_spoVG RBS & BB_mrgApromoterrbs<br />
Failed transformation<br />
Alyssa: Made Excel template for ligation calculations. Phosphorylated, annealed, and ligated purchased oligonucleotides:<br />
mrgA, mntH, spoVG1, spoVG2<br />
<br />
Malinka: Phosphorylated and annealed mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS<br />
Matt: Previously we determined that both Tet resistant and unresistant cells grow in 1x Tet liquid culture but die in 10x liquid culture. Our purpose is to determine a Tet concentration between these two extremes where Tet resistant culture lives and Tet unresistant culture does not. Using strains: PSB1AT3 in DH5alpha and PUC19 in DH5alpha at concentration of 0X, 2.5X, 3X, 3.5x, and 4X Tet<br />
(1X Tet= 50ug/ml, stock is at 100X) Culture in 5ml of LB, inoculate from picking plates. At 2.5X PSB1At3 DH5alpha grew but PUC19 DH5alpha did not. Above this concentration nothing grew.<br />
<br />
<br />
<br />
8/11 Malinka: : Transformed and plated mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS (all on pSB1AC3).<br />
Alyssa: Ligated and transformed oligos of mntH promoter and mrgA promoter. Transformed and plated RBS and mnth promoter biobricks in DH5 cells<br />
<br />
<br />
<br />
8/12 Xing: Test transformation protocol and competency of cells <br />
Cells competent and transformation worked with pUC19<br />
Alyssa: CdCl2 growth curve plate design<br />
<br />
<br />
8/13 Xing: Tranformation of mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
Successful transformation<br />
Ben: Inoculated spoVG transformants for mini-prepping<br />
Malinka: Cultured amyE parts and made amp plates.<br />
Alyssa:<br />
Redid CdCl2 growth curves redo the CdCl2 growth curve experiment from 7/18, which resulted in biphasic growth curves and growth of mntR when exposed to Cd. (This should not have happened due to its extreme Cd sensitivity.)<br />
--Results:<br />
No errors <br />
111 runs<br />
Biphasic growth curves for all strains, though not as pronounced as 1st growth curve exp.<br />
mntR shows more growth than WT, even at 5uM Cd <br />
<br />
<br />
<br />
8/14 Xing: Inoculate cultures from transformant plates for mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
PAGE purification of mrgApromrbs<br />
Alyssa:<br />
Made new 1000X Tet stock and measured concentration of PSB1K3-- to make a more potent Tet stock soln. and measure concentration of a biobrick backbone for calculation purposes in other experiments and procedures (such as ligation).<br />
Analyzed data from 8/13 growth curves on Excel<br />
Cut and dephosphorylated psB1K3 backbone<br />
<br />
<br />
8/15 Xing: Digest and gel analysis of spoVG, BBa_K143001, BBa_K143002<br />
spoVG migrates with expected mobility, BBa_K143001 & 2 do not migrate as expected<br />
<br />
Miniprep mntH, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
8/19 Alyssa: Digestion of mntH, mnrA promoter, mrgA + mrgA rbs, and mrgA rbs<br />
Matt: digested Biobricks in Preparation for 3A Ligation and Gels.<br />
<br />
<br />
8/20 Alyssa:<br />
Phosphorylization, annealing, and ligation of mntH, mrgA, spoVG, yvgW, and amyE locus biobricks-- to make biobrick constructs out of the oligos that we ordered<br />
Performed ligation calculations for mrgA promoter, mntH promoter, spoVG, amyE locus, and yvgW promoter<br />
<br />
<br />
<br />
8/30 Malinka: Made Kan plates.<br />
<br />
8/31 Xing: Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/1 Matt: Inoculated mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, and yvgW/spoVG to prepare for a miniprep. The plates he inncoculated from: No L 50ul control looks almost completely clear. No L 250ul control has very small speckling on surface. Both 50ul and 250ul no I controls grew dozens of singles. PUC19 grew. H20 control is clear. All samples grew.<br />
<br />
<br />
9/2 Xing: Miniprep mntH/mrgA rbs, mntH/spoVG, mrgA/mrgArbs, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG<br />
<br />
9/3 Alyssa: Digested mntH/ mrgArbs, mntH/ spoVG rbs, YFP, and GFP<br />
<br />
<br />
9/4 Xing: Ligation of mntH-mrgA/YFP, mntH-spoVG/YFP, mrgAprom-mrgArbs/GFP and transformation<br />
<br />
9/7 Xing: Miniprep cultures of mntH/spoVG/YFP, mntH/mrgArbs/YFP, mrgAprom/spoVG, yvgW/mrgArbs, yvgW/spoVG <br />
<br />
9/8 Malinka: Transforming and plating promoter + RBS bricks (yvgW and mrgAprom with spoVG or mrgArbs).<br />
Matt tested to see if backbones took up desired sequences (mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs) by running a gel. Plasmids cut with E and P show slightly smaller bands than those cut with just E on both gels. Wells 6 and 7 corresponding to mrgApmrgA are very light.<br />
<br />
<br />
9/9 Xing: Send constructs for sequencing<br />
mrgA promoter, mrgA rbs proper sequence. mntH, yvgW, spoVG inserted inverted into plasmid.<br />
<br />
9/11: Miniprep and digestion of yvgW and mrgA constructs<br />
<br />
9/13 Xing: Digestion and Gel Analysis of mntH, yvgW, spoVG parts <br />
spoVG 6:1 2,3 migrate as expected, send for sequencing<br />
Send spoVG 6:1 2,3 for sequencing<br />
Both samples proper sequence<br />
<br />
9/15: Alyssa Miniprepped 9/14 inoculated cultures—yvgW, mntH, spoVG<br />
<br />
<br />
9/16 Xing: Digestion and Gel Analysis of mntH, yvgW<br />
No samples appear linearized<br />
Matt prepared competent DH5alpha E Coli cells. Grew two 50ml cultures to .35 OD600 and .37 OD600.<br />
Pellets re-suspended fine. About 80-85 100ul aliquots of competent cells were stored in -80C.<br />
<br />
9/21 Xing: Ligation of mntH, yvgW inserts into pSB1AC3 backbones<br />
pUC19 control shows colonies, all other plates blank<br />
<br />
9/22 Matt checked to see that the newly transformed plates from 9/21 did not grow, so he inncoulated only from the old 8/13 and 7/22 plates. Taking from colonies that do not appear to have been sampled: 9 colonies from yvgW 6:1 buc 7/22 3 colonies from mntH 6:1 buc 8/13 3 colonies from mntH 6:1 buc 8/13 3 colonies from mntH 6:1 buc 8/13<br />
<br />
<br />
9/24 Malinka: : Digestion (with X) and Gel Analysis of mntH, yvgW parts to determine which promoter biobricks were good and should be sequenced.<br />
Alyssa: Miniprepped mntH, yvgW<br />
<br />
9/25 Malinka: : Preparing mntH, yvgW biobricks for sequencing.<br />
<br />
9/28 Xing: Digestion and Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/29 Malinka: Culturing (mntH and yvgW) + RBS (spoVG) biobricks.<br />
<br />
9/30 Xing: Preparing promoter rbs constructs for sequencing<br />
yvgW/spoVG 1 9/30 displays proper sequence<br />
<br />
10/3 Ben<br />
Digested yvgW/spoVG, CFP, and pSB1AC3 (rfp insert)<br />
Ligated yvgW/spoVG to CFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
<br />
10/6 Matt innoculated from 8/31yvgW/mrgArbs and 10/5 yvgW/spovG/cfp. 10/5 yvgW/spovG/cfp grew on chlor all colonies white many colonies. The chlor controls were clear.<br />
<br />
<br />
10/10 Malinka: Transforming and plating yvgW/spoVG/GFP biobricks and yvgW/spoVG biobricks <br />
Alyssa: <br />
Digested yvgW/spoVG, GFP, PSB1AC3<br />
Ligated promoter/rbs and GFP to backbone<br />
<br />
10/11 Matt streaked 2 plates of B Subtillis Wild Type (CU1065) from frozen permanent stock to grow up for transformation. Nothing grew on either plate, the permanents must be bad. <br />
<br />
10/12 Malinka: 3 way ligation to construct mrgAprom/mrgArbs parts. Re-digesting plasmids to construct yvgW/spoVG/GFP parts<br />
Alyssa: Plated mrgA/ mrgA rbs on Kan plates<br />
<br />
<br />
10/16 Xing: Preparation of Kan, Chlor, LB plates<br />
<br />
<br />
10/17 Xing: Preparing samples for registry<br />
Ben<br />
Digested yvgW/spoVG, GFP, and pSB1AC3 (rfp insert)<br />
Ligated yvgW/spoVG to GFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
Malinka: Preparing samples for registry. Re-transforming and plating yvgW/spoVG/GFP<br />
<br />
10/20: Alyssa :<br />
Miniprepped yvgW/spoVG/GFP on Chlor—pink pellets, so did not miniprep in full to obtain DNA<br />
Inoculated six tubes of colonies plated on 10/17—yvgW/spoVG/GFP on chlor plate.</div>Bammarata89http://2009.igem.org/Team:Cornell/NotebookTeam:Cornell/Notebook2009-10-22T03:21:28Z<p>Bammarata89: </p>
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<br />
6/26/09<br />
1. Alyssa and Matt—made chloramphenicol, kanamycin, and tetracycline antibiotic stock solutions for use in agar plates. See “Protocols” folder for antibiotic-making protocol.<br />
<br />
<br />
Antibiotic Volume (mL): Stock (mg/mL): Amnt use (g): Solvent<br />
chloramphenicol 15 34(200x) 0.510 EtOH<br />
kanamycin 15 10(200x) 0.150 H20<br />
tetracycline 50 5(200x) 0.250 EtOH<br />
<br />
• Comments: <br />
o Please note that tetracycline does NOT fully dissolve in 100% EtOH—will see a yellow powder in a clear solution.<br />
o For all stock solutions of antibiotics dissolved in H20: “sterilize by filtration through a 0.22um filter (Sambrook and Russel)”<br />
<br />
2. Xing—PCR cloning of GFP+ into Biobrick standard A<br />
<br />
6/29/09<br />
<br />
1. Malinka and Ben—Spread antibiotics on agar plates for ccdb amplification. Created a table for the antibiotics we were going to use and then top-spread antibiotics onto agar plates. <br />
<br />
• Comments:<br />
o The color code for the antibiotics: <br />
Amp – Black<br />
Chlor – Red<br />
Kan – Green<br />
Tet – Blue<br />
o Table they came up with is Ben’s notebook entry for the day. Results are summarized as the following: <br />
--pSB1AK3 had colonies on each plate<br />
--water plates had no colonies, except for amp plates<br />
--pSB1AC3 and pSB1AT3 had no colonies, except for on the amp plates<br />
2. Xing-- Agarose Gel Electrophoresis Analysis of GFP+ PCR Product to analyze products of PCR of GFP+<br />
<br />
<br />
Lanes 1 2 3 4 5 6 7 8<br />
PFx 1ng 5λ <br />
PFx 10ng 5λ <br />
PFx 100ng 5λ <br />
Pfu 1ng 5λ <br />
Pfu 10ng 5λ <br />
Pfu 100ng 5λ <br />
pGFP+ 1 5λ <br />
100bp ladder Fermentas 5λ<br />
6X Loading dye 2λ 2λ 2λ 2λ 2λ 2λ 2λ <br />
Total Volume 7λ 7λ 7λ 7λ 7λ 7λ 7λ 5λ<br />
<br />
• Comments:<br />
o The rxns using Pfx display product at the expected mobility ~750bp with increasing yields following increasing amount of template plasmid used. The rxns treated with Pfu displayed no product.<br />
<br />
6/30/09<br />
<br />
1. Xing—PCR Amplification of yvgY promoter & GFP+. The goal was to PCR Amplify yvgY promoter from chromosome into BioBrick standard and repeat GFP+ amplification since we used 10X more primer in the previous experiment than we should have.<br />
<br />
2. Malinka and Ben—extracted iGEM backbone plasmids from kit (wells 11c for pSB1AC3, 13e for pSB1AK3, and 15c for pSB1AT3) and transformed them. Also grew B. subtilis strains to make frozen permanents. <br />
• Comments:<br />
o Ben's plates (for pSB1AK3) had various numbers of colonies<br />
o Malinka’s plates (for pSB1AC3 and pSB1AT3)<br />
--Nothing grew on the 50 ul of only Tet or Chlor<br />
--Nothing grew in the 50 ul or 250 ul of the Tet and amp and the Chlor and <br />
Amp<br />
--No colonies on water plates<br />
--Some cells grew on the only amp plates. Thus amp not very effective, and <br />
there was a large “background”<br />
7/1 <br />
o Xing; Agarose Gel Electrophoresis of GFP+/yvgY promoter PCR Products<br />
GFP+ bands display expected mobility, with greater yield in the 1ng rxn than the previous rxn. No apparent product in rxns amplified by Pfu. yvgY shows up faintly for rxns using Pfx and Pfu with expected mobility <100bp. <br />
o Alyssa and Matt: Growth Curve Characterization of B Subtilis at Different Culture Scales--To characterize the growth curves of B Subtilis in both ml scale tubes and in microtiter plates by measuring optical density at set intervals during growth. (By finding when the strains enter logarithmic growth phase, we can better design our B-galactosidase assay protocol.) Calculations assume midlog phase.<br />
o Ben: Inoculating pSB1AK3 & re-plating pSB1AC3 and pSB1AT3. Results: Inoculating pSB1AK3<br />
-All tubes contain cells…SUCCESS! Re-plating pSB1AC3 and pSB1AT3<br />
-None of the water plates grew cells, except for the tet lower concentration plate<br />
-The supposed plasmid containing cells also did not grow, except for some background on the lower concentration tet plate.<br />
o Malinka: Replating plasmids and making frozen permanents-- Replate the pSB1AC3 and pSB1AT3 and do frozen permanents<br />
--Results: Plasmids didn’t grow, lawn on water<br />
<br />
7/2 <br />
o Xing: Gel Electrophoresis of yvgY promoter PCR rxn with primers<br />
Compare yvgY PCR rxns with primers to confirm or refute presence of product<br />
Primers do not show up on gel, there is a band below 100bp for the PCR rxns. The expected product size is ~120 bp. The <100 band could be due to primer dimerization. Run a primer titration down to 50nM primer to check next time.<br />
o Ben: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain. Results: Re-transforming pSB1AC3 and pSB1AT3-No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates). plating ccdB-resistant strain -2 lawns formed<br />
o Malinka: Re-transforming pSB1AC3 and pSB1AT3 and plating ccdB-resistant strain<br />
--Results: -No plates contained cells, except for water amp, and puc19 (both had empty circle inside, may be problem with plates) plating ccdB-resistant strain-2 lawns formed<br />
<br />
7/3 <br />
o Xing: Preparation of Chemically Competent E.Coli. Part II<br />
o Xing: Gel Analysis PCR of yvgY and yvgW promoters primer titration; Run agarose gel electrophoresis to analyze PCR rxns-- yvgY rxns display the same band <100bp but appears less intense for .1ng/uL primer rxn and even less for .05ng/uL primer rxn, supporting the hypothesis that these are primer dimerization products. yvgW does not display any products. Possibility of broken chromosomal DNA? Extract Bacillus DNA next time.<br />
o Xing: PCR Amplification of yvgY and yvgW promoter-- PCR amplify yvgY and yvgW promoter into BB standard and titrate primers to see if proper product forms.<br />
o Xing: Prepare Kanamycin and Tetracycline Plates<br />
o Ben: Preparation of Chemically Competent E.Coli.<br />
o Malinka: Prepare Chloramphenicol and Ampicillin Plates, and grow competent cells<br />
<br />
7/6<br />
o Ben and Malinka: Checking competency of cells/ if plates are working (by Malinka). Bad results, redo experiment<br />
7/7<br />
o Ben: Re-re-transforming cells (LOL). Transform (hopefully) pSB1AC3 and pSB1AT3 in ccdB-resistant and DH5a strains.<br />
--Results: -Tet plates (including water and both pSB1AT3 plates) contained lawns, reason unkown<br />
-Chlor plates came out as predicted, nothing on water plates, dozens of colonies on 250uL plate, <10 on 50uL plate, but colonies were not red still need to view them under UV light<br />
-water controls were clean, except for Tet, as said above<br />
-pUC19 1:10 dilution had <10 colonies, 1:100 had none visible replating at 1:1 to check competent cells’<br />
o Malinka: Replating plasmid backbones-- Playing pSB1AK3 and pSB1AC3 on new antibiotic plates<br />
--Results: pSB1AT3 (ccdb) – Proportional amounts of single colonies had grown on my 50 and 250 ul plates. However a similar amount of colonies had grown on the water too. <br />
<br />
pSB1AT3 (DH5a) – Ben had lawns on all his Tet plates.<br />
<br />
pSB1AC3 (ccdb) – I had nothing on all 3 of my chlor plate.<br />
<br />
pSB1AC3 (DH5a) – Proportional amounts of single colonies had grown on his 50 and 250 ul plates. Nothing had grown on his water. The cells weren’t red like we expected them to be but that was probably because they were too small.<br />
<br />
Water (1:1000) (ccdb and DH5a) – We had lots of single colonies on both our plates. They looked similar.<br />
<br />
pUC19 (ccdb) - <br />
<br />
pUC19 (DH5a) - <br />
<br />
<br />
7/8<br />
o Xing: PCR yvgY promoter & yvgW temperature using a temperature gradient<br />
o Xing: Preparation of mntR frozen permanents<br />
o Ben: Innoculating pSB1AC3 and pSB1AT3 (rfp versions) to see if we have transformed backbones. <br />
--Results: Chlor plasmids had growth, reddish in tube!-Tet did not grow, as expected<br />
o Malinka: culture backbone from the plates done yesterday<br />
--Results: All of Ben’s pSB1AC3s grew and turned red. Only 1 of Malinka’s pSB1AT3s grew<br />
o Alyssa: Preparing B. subtilis cultures exposed to ZnCl2 for practice LacZ Assay-- To try out the B-Gal assay protocol mentioned on page 12 of Helmann’s “Genetic and physiological responses of B. subtilis to metal ion stress” paper<br />
--Results: Whoa! Heavy metals/ growth retardation in action! According to the last OD600 readings, ZnCl2 seems to significantly (and negatively) affect the growth rates of the B. subtilis strains exposed to the highest concentrations of metal solution.<br />
<br />
7/9 <br />
o Xing: Agarose Gel Electrophoresis of PCR yvgY yvgW temperature gradient to analyze PCR products-- yvgY rxn still forms primer dimmers, though concentration seems to decrease with increasing temperature. No products for yvgW rxn.<br />
o Matt and Alyssa: Designed Zinc growth curve plate<br />
o Ben: Mini-prep pSB1AC3 (rfp) and pSB1AK3 (ccdB)<br />
7/10<br />
o Xing: Agarose Gel Electrophoresis of Temperature Gradient to analyze PCR products<br />
o Xing: PCR temperature gradient of yvgY & yvgW-- PCR yvgY & yvgW promoters with positive controls.<br />
7/13<br />
o Malinka: Digesting the plasmid backbones PSB1AC3 and PSB1AK3<br />
<br />
7/14<br />
o Xing: Gel electrophoresis of varying [Mg2+] PCR<br />
o Xing: PCR Amplification of yvgY & yvgW w/ varying [Mg2+]-- Vary [Mg2+] in PCR rxn to see if products form<br />
o Malinka: ran gels for backbones<br />
7/15<br />
o Matt: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
o Malinka: Digestion - Removing inserts from the plasmid backbones PSB1AC3, PSB1AK3, PSB1AT3<br />
--Results: <br />
o <br />
7/16<br />
o Ben: DNA electrophoresis of doubly digested (X & S) backbones and GFP+<br />
7/17<br />
o Xing: PNK treatment and annealing of complementary primers-- PNK treat primers to phosphorylate ends for ligation/anneal complementary primers<br />
o Alyssa: CdCl2 soln growth curve plate design for use in TECAN machine<br />
7/20<br />
o Ben: Check of pSB1AK3 ccdB-gene effectiveness through transformation in DH5a<br />
--Results: -waters were blank-puc19 had a few cells -sSB1AK3 plates both transformed, meaning the ccdB gene is broken, since the cells should have died<br />
o Malinka: Dephosphoralating and purifying backbone plasmids PSB1AC3, PSB1AK3, PSB1AT3<br />
7/21<br />
o Matt and Alyssa: Cadmium and Zinc Beta Galactosidase Assay-- To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion.<br />
o Ben: Ligation of promoters and GFP+<br />
o Malinka: Creating GFP and promoter biobricks—yvgW and yvgY<br />
--Results: Based on yesterdays Tet plate results, we found that the Tet plates work. But the ccdb psb1AK3 does not work so we have only pSB1AC3 and pSB1AT3 as backbones.<br />
7/22<br />
o Ben: Transformation of ligations yvgW and GFP+<br />
o Malinka: Redigestion - Removing inserts from the plasmid backbones<br />
Results: Plated Ligated Biobricks <br />
<br />
Controls:<br />
The pUC19 on amp showed reasonable growth and the water on amp had nothing which meant that there was no background on the amp plates. As we still got good growth for pUC19, this showed that DH5 cells were alive and competent. The water on the Chlor plater had no growth. <br />
<br />
The no insert and no enzyme ligation mixtures showed no growth.<br />
<br />
yvgW and yvgY:<br />
There was little growth on the 2:1 ratio plates for yvgW and yvgY (maybe 5 colonies). There was more growth on the 6:1 ratio plates (maybe 15 colonies) – we plan to use 6:1 ratio from now on.<br />
<br />
GFP:<br />
There was no growth on any of the GFP biobrick plates (2:1, 4:1 or 6:1).<br />
<br />
Undigested plasmid (pSB1AC3) would have shown up as red cells on the plate. However from previous experience, we knew that 24 hours of incubation wasn't enough for the cells to have grown large enough or long enough to be visibly red. So we weren't sure, of the plates with growth in them, which cells actually had been transformed with our biobricks as opposed to the undigested plasmid. <br />
<br />
7/23<br />
o Ben: Transformation of pSB1K3 and pSB1T3 backbones<br />
--Results: -Kan plates produced colonies (slightly red) -waters had nothing (except tet plates bad plates) -pUC19 had colonies -tet plates all had lawns bad plates<br />
o Malinka: To run gels on the newly digested plasmid backbones and cut GFP to make sure we had got them right. To dephosphorylate the cut backbones and then purify them so that we had enough vector to ligate with our inserts later.<br />
--Results: The gels looked good for Chlor, Tet and Kan. I was even able to see a the insert for Kan very low in the gel (it was very faint) – exactly where the iGEM gel showed it to be.<br />
<br />
I got nothing for the cut and uncut GFP. Ben had used uncut GFP from the same tube a few days ago and got results but he used 1 ul which is (600-700 ng) so perhaps I had too little. We need to do this again with more cut and uncut GFP.<br />
<br />
<br />
o Xing – To culture tubes with colonies from our transformed biobricks (with promoters). Also to culture 1 tube with pSB1AC3 (with RFP) because we were going to use RFP (and GFP) for our project. <br />
<br />
o Ben and Matt – To make new Chlor and Tet plates and transform cells with new backbone plasmid from the kit (pSB1T3 & pSB1K3) in case our next backbone doesn't work. <br />
7/24<br />
o Cadmium and Zinc Beta Galactosidase Assay in tubes: To characterize the effects of Cadmium and Zinc concentration on the promoter for the CadA-lacZ fusion. Couldn’t access spectrometer when the assay was complete, so tubes were stopped when they showed a clear yellow color and left to sit out over the weekend. On Monday all remaining tubes were stopped and OD420, OD550, and OD600 were measured in the spec.<br />
o Malinka: Test--Digesting the pSB1AC3 and pSB1AT3 using 15 minute method and digesting purified GFP+<br />
--Results: The 15 minute digest of pSB1AC3 and pSB1AT3 showed the same results as the digests of the same plasmids done overnight.<br />
<br />
We saw the uncut GFP but did not see the cut GFP. We think it’s because the GFP+ wasn’t purified.<br />
<br />
<br />
<br />
7/24<br />
o Xing: Purification of GFP+ PCR Products<br />
<br />
7/27<br />
o Xing; Purification of Additional GFP+ PCR Products<br />
o Alyssa and Malinka: Digesting GFP+ and running gels on it-- To test pure cut GFP+ digested for 6 hours, cut and then test GFP+ digested for 15 minutes, cut and then test new backbones, and cut and then test biobrick parts with promoters.<br />
7/29<br />
o Ben: Mini-prep of GFP (E0040), CFP (E0020), and YFP (E0030)<br />
o Alyssa: designed primers to extract constitutive mrgA promoter from Helmann strain of B. sub.<br />
7/30<br />
o Alyssa and Xing: Digest pSB1K3 & ligate GFP+ part onto digest--Digestion of assembly vector pSB1K3, dephosphorylation & ligation of GFP+ onto assembly vector. <br />
o Ben: Digestion of GFP CFP and YFP-- Appropriate bands located in all wells: perfect!<br />
o <br />
o <br />
<br />
7/31<br />
o Xing: Transformation of GFP+ Ligation and selection with Kanamycin<br />
o Xing: Transformation of pSB1AC3 using Amp selection--1 Colony for 50uL pSB1AC3<br />
~ 10 Colonies for 250 uL pSB1AC3, no colonies for H2O, pUC19 250 uL 0-5 colonies.<br />
o Alyssa and Ben: Ligation of assembly vector PSBK3 to GFP+ to create GFP+ biobrick parts<br />
<br />
8/3<br />
Xing: Digest & gel analysis of yvgW, yvgY, GFP+ BB parts<br />
- No GFP+ parts, incomplete digestion for yvgW, yvgY parts.<br />
Malinka: Mini-prepped GFP+ biobricks and pSB1AC3<br />
8/4 Xing: Digest & gel analysis of yvgW, yvgY BB parts<br />
- Mobility as expected for for yvgW 2:1 1 and yvgY 6:1 3<br />
Malinka: Transformed and plated pSB1AT3 (rfp and ccdb) from the kit.<br />
<br />
8/5 Xing: Testing effectiveness of tetracycline plates<br />
- Lawn formed on all tet plates<br />
Malinka: Cultured pSB1AT3 (DH5) cells, and yvgY (on pSB1AC3), yvgW (on pSB1AC3) and GFP+ (on pSB1AK3) biobricks.<br />
<br />
Digest & gel analysis of yvgW 2:1 1, yvgY 6:1 3, GFP+ 2:1 50 BB parts<br />
- yvgW 2:1 1 sample with expected mobility, yvgY 6:1 3 sample incompletely digested, GFP+ 2:1 50 sample not as expected<br />
<br />
8/10 Xing: Transformation of BB_spoVG RBS & BB_mrgApromoterrbs<br />
Failed transformation<br />
Alyssa: Made Excel template for ligation calculations. Phosphorylated, annealed, and ligated purchased oligonucleotides:<br />
mrgA, mntH, spoVG1, spoVG2<br />
<br />
Malinka: Phosphorylated and annealed mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS<br />
<br />
8/11 Malinka: : Transformed and plated mrgA promoter, mrgA RBS, mrgA promoter-RBS, mntH promoter and spoVG RBS (all on pSB1AC3).<br />
Alyssa: Ligated and transformed oligos of mntH promoter and mrgA promoter. Transformed and plated RBS and mnth promoter biobricks in DH5 cells<br />
<br />
<br />
<br />
8/12 Xing: Test transformation protocol and competency of cells <br />
Cells competent and transformation worked with pUC19<br />
Alyssa: CdCl2 growth curve plate design<br />
<br />
<br />
8/13 Xing: Tranformation of mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
Successful transformation<br />
Ben: Inoculated spoVG transformants for mini-prepping<br />
Malinka: Cultured amyE parts and made amp plates.<br />
Alyssa:<br />
o Redid CdCl2 growth curves redo the CdCl2 growth curve experiment from 7/18, which resulted in biphasic growth curves and growth of mntR when exposed to Cd. (This should not have happened due to its extreme Cd sensitivity.)<br />
--Results:<br />
• No errors <br />
• 111 runs<br />
• Biphasic growth curves for all strains, though not as pronounced as 1st growth curve exp.<br />
• mntR shows more growth than WT, even at 5uM Cd <br />
<br />
<br />
<br />
8/14 Xing: Inoculate cultures from transformant plates for mntH promoter, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
PAGE purification of mrgApromrbs<br />
Alyssa:<br />
o Made new 1000X Tet stock and measured concentration of PSB1K3-- to make a more potent Tet stock soln. and measure concentration of a biobrick backbone for calculation purposes in other experiments and procedures (such as ligation).<br />
o Analyzed data from 8/13 growth curves on Excel<br />
o Cut and dephosphorylated psB1K3 backbone<br />
<br />
<br />
8/15 Xing: Digest and gel analysis of spoVG, BBa_K143001, BBa_K143002<br />
spoVG migrates with expected mobility, BBa_K143001 & 2 do not migrate as expected<br />
<br />
Miniprep mntH, mrgA promoter, mrgA rbs, BBa_K143001, BBa_K143002<br />
<br />
8/19 Alyssa: Digestion of mntH, mnrA promoter, mrgA + mrgA rbs, and mrgA rbs<br />
<br />
8/20 Alyssa:<br />
o Phosphorylization, annealing, and ligation of mntH, mrgA, spoVG, yvgW, and amyE locus biobricks-- to make biobrick constructs out of the oligos that we ordered<br />
o Performed ligation calculations for mrgA promoter, mntH promoter, spoVG, amyE locus, and yvgW promoter<br />
<br />
<br />
<br />
8/30 Malinka: Made Kan plates.<br />
<br />
8/31 Xing: Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/2 Xing: Miniprep mntH/mrgA rbs, mntH/spoVG, mrgA/mrgArbs, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG<br />
<br />
9/3 Alyssa: Digested mntH/ mrgArbs, mntH/ spoVG rbs, YFP, and GFP<br />
<br />
<br />
9/4 Xing: Ligation of mntH-mrgA/YFP, mntH-spoVG/YFP, mrgAprom-mrgArbs/GFP and transformation<br />
<br />
9/7 Xing: Miniprep cultures of mntH/spoVG/YFP, mntH/mrgArbs/YFP, mrgAprom/spoVG, yvgW/mrgArbs, yvgW/spoVG <br />
<br />
9/8 Malinka: Transforming and plating promoter + RBS bricks (yvgW and mrgAprom with spoVG or mrgArbs).<br />
<br />
9/9 Xing: Send constructs for sequencing<br />
mrgA promoter, mrgA rbs proper sequence. mntH, yvgW, spoVG inserted inverted into plasmid.<br />
<br />
9/11: Miniprep and digestion of yvgW and mrgA constructs<br />
<br />
9/13 Xing: Digestion and Gel Analysis of mntH, yvgW, spoVG parts <br />
spoVG 6:1 2,3 migrate as expected, send for sequencing<br />
Send spoVG 6:1 2,3 for sequencing<br />
Both samples proper sequence<br />
<br />
9/15: Alyssa Miniprepped 9/14 inoculated cultures—yvgW, mntH, spoVG<br />
<br />
<br />
9/16 Xing: Digestion and Gel Analysis of mntH, yvgW<br />
No samples appear linearized<br />
<br />
9/21 Xing: Ligation of mntH, yvgW inserts into pSB1AC3 backbones<br />
pUC19 control shows colonies, all other plates blank<br />
<br />
9/24 Malinka: : Digestion (with X) and Gel Analysis of mntH, yvgW parts to determine which promoter biobricks were good and should be sequenced.<br />
Alyssa: Miniprepped mntH, yvgW<br />
<br />
9/25 Malinka: : Preparing mntH, yvgW biobricks for sequencing.<br />
<br />
9/28 Xing: Digestion and Ligation of mntH/mrgArbs, mntH/spoVG, mrgAprom/mrgARBS, mrgA/spoVG, yvgW/mrgArbs, yvgW/spoVG, and transformation.<br />
<br />
9/29 Malinka: Culturing (mntH and yvgW) + RBS (spoVG) biobricks.<br />
<br />
9/30 Xing: Preparing promoter rbs constructs for sequencing<br />
yvgW/spoVG 1 9/30 displays proper sequence<br />
<br />
10/3 Ben<br />
o Digested yvgW/spoVG, CFP, and pSB1AC3 (rfp insert)<br />
o Ligated yvgW/spoVG to CFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
<br />
10/10 Malinka: Transforming and plating yvgW/spoVG/GFP biobricks and yvgW/spoVG biobricks <br />
Alyssa: <br />
o Digested yvgW/spoVG, GFP, PSB1AC3<br />
o Ligated promoter/rbs and GFP to backbone<br />
<br />
<br />
10/12 Malinka: 3 way ligation to construct mrgAprom/mrgArbs parts. Re-digesting plasmids to construct yvgW/spoVG/GFP parts<br />
Alyssa: Plated mrgA/ mrgA rbs on Kan plates<br />
<br />
<br />
10/16 Xing: Preparation of Kan, Chlor, LB plates<br />
<br />
<br />
10/17 Xing: Preparing samples for registry<br />
Ben<br />
o Digested yvgW/spoVG, GFP, and pSB1AC3 (rfp insert)<br />
o Ligated yvgW/spoVG to GFP in pSB1AC3 following Ginkgo Bioworks protocol<br />
Malinka: Preparing samples for registry. Re-transforming and plating yvgW/spoVG/GFP<br />
<br />
10/20: Alyssa :<br />
o Miniprepped yvgW/spoVG/GFP on Chlor—pink pellets, so did not miniprep in full to obtain DNA<br />
o Inoculated six tubes of colonies plated on 10/17—yvgW/spoVG/GFP on chlor plate.</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T03:00:33Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="900px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Project/Background|Background]]<br />
!align="center"|[[Team:Cornell/Project/Chassis|Chassis]]<br />
!align="center"|[[Team:Cornell/Project/Design|Design]]<br />
!align="center"|[[Team:Cornell/Project/Standardization|Standardization]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
= Team Description =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
= Advisors =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
= Undergrads =<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:CornellTeam:Cornell2009-10-22T02:59:49Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="900px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Project/Background|Background]]<br />
!align="center"|[[Team:Cornell/Project/Chassis|Chassis]]<br />
!align="center"|[[Team:Cornell/Project/Design|Design]]<br />
!align="center"|[[Team:Cornell/Project/Standardization|Standardization]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
=Project Abstract=<br />
[[Image:CUTeam.JPG|right|500px]]<br />
Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture, Itai-Itai disease, infertility, damage to the central nervous and immune systems, cancer development and ultimately death. For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. Of late, cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems especially in developing countries where there is little regulation of industrial waste products. The goal of our project is to create a low-cost bio-sensor by using the metal ion homeostasis system of Bacillus subtilis to quantify environmental levels of cadmium in drinking water. Since B. subtilis has genetic elements that naturally respond to different concentrations of metal ions, it is an excellent system to serve as our bio-sensor chassis.</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:44:34Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
[[Image:CornellTeam.JPG|500px|right]]<br />
<br />
= Team Description =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
= Advisors =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
= Undergrads =<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:42:16Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
[[Image:CornellTeam.jpg]]<br />
<br />
= Team Description =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
= Advisors =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
= Undergrads =<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/File:CornellTeam.JPGFile:CornellTeam.JPG2009-10-22T02:39:25Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:38:18Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
= Team Description =<br />
[[Image:CUTeam.jpg|300px|right]]<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
= Advisors =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
= Undergrads =<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:35:26Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
= Team Description =<br />
[[Image:CUTeam.jpg|300px|right]]<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
= Advisors =<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:31:28Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
[[Image:CUTeam.jpg|300px|right]]<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/File:CUTeam.JPGFile:CUTeam.JPG2009-10-22T02:31:14Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Team.JPGFile:Team.JPG2009-10-22T02:29:02Z<p>Bammarata89: uploaded a new version of "Image:Team.JPG"</p>
<hr />
<div></div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:27:48Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
[[Image:Team_photo.jpg|300px|right]]<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:25:33Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
<br />
<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:24:01Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/File:Xing.jpgFile:Xing.jpg2009-10-22T02:23:33Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Hellmann.jpgFile:Hellmann.jpg2009-10-22T02:22:22Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:21:31Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt: Professor, Food Science<br />
Image:Maki_Inada.jpg|Maki Inada: Adjunct Professor, Molecular Biology and Genetics<br />
Image:Hellmann.jpg|John D. Helmann: Professor, Microbiology<br />
Image:XilingShen.jpg|Xiling Shen: Assistant Professor, Electrical and Computer Engineering<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing_Xiong.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:17:13Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt<br />
Image:Maki_Inada.jpg|Maki Inada<br />
Image:XilingShen.jpg|Xiling Shen<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing_Xiong.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:13:26Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt<br />
Image:Maki_Inada.jpg|Maki Inada<br />
Image:XilingShen.jpg|Xiling Shen<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing_Xiong.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
<br />
<br />
<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka was born in Sri Lanka. He doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:12:36Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt<br />
Image:Maki_Inada.jpg|Maki Inada<br />
Image:XilingShen.jpg|Xiling Shen<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
[[Image:Xing_Xiong.jpg|100px|right]]<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
[[Image:Matthew_Hall.jpg|100px|right]]<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
== Malinka Walaliyadde ==<br />
[[Image:Malinka.jpg|100px|right]]<br />
Malinka doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
[[Image:Kevin_Cheng.jpg|100px|right]]<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:04:18Z<p>Bammarata89: /* Team Members */</p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
= Advisors =<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt<br />
Image:Maki_Inada.jpg|Maki Inada<br />
Image:XilingShen.jpg|Xiling Shen<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
== Malinka Walaliyadde ==<br />
Malinka doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T02:03:26Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
'''Advisors:'''<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Carl_Batt.jpg|Carl Batt<br />
Image:Maki_Inada.jpg|Maki Inada<br />
Image:XilingShen.jpg|Xiling Shen<br />
<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
== Malinka Walaliyadde ==<br />
Malinka doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/File:XilingShen.jpgFile:XilingShen.jpg2009-10-22T01:59:50Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Matthew_Hall.jpgFile:Matthew Hall.jpg2009-10-22T01:58:34Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Malinka.jpgFile:Malinka.jpg2009-10-22T01:57:49Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Maki_Inada.jpgFile:Maki Inada.jpg2009-10-22T01:56:41Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T01:56:01Z<p>Bammarata89: </p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
'''Advisors:'''<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Team_member_1.png|Team member 1<br />
Image:Team_member_2.png|Team member 2<br />
Image:Team_member_3.png|Team member 3<br />
Image:Team_member_4.png|Team member 4<br />
Image:Team_member_5.png|Team member 5<br />
Image:Team_member_6.png|Team member 6<br />
Image:Team_member_6.png|Team member 7<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata == <br />
[[Image:Ben_Cammarata.jpg|100px|right]]<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
[[Image:Alyssa Henning.jpg|175px|right]]<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
== Malinka Walaliyadde ==<br />
Malinka doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/File:Kevin_Cheng.jpgFile:Kevin Cheng.jpg2009-10-22T01:55:12Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Carl_Batt.jpgFile:Carl Batt.jpg2009-10-22T01:54:23Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T01:48:07Z<p>Bammarata89: /* Bernard “Big Ben” Cammarata */</p>
<hr />
<div>[[Image:CUGEM_Banner.png|center|800px]]<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:10px"<br />
|-<br />
|rowspan="3"|<br />
<br />
'''Advisors:'''<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' John D. Helmann''': Professor, Microbiology<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
|<br />
<gallery><br />
Image:Team_member_1.png|Team member 1<br />
Image:Team_member_2.png|Team member 2<br />
Image:Team_member_3.png|Team member 3<br />
Image:Team_member_4.png|Team member 4<br />
Image:Team_member_5.png|Team member 5<br />
Image:Team_member_6.png|Team member 6<br />
Image:Team_member_6.png|Team member 7<br />
</gallery><br />
|-<br />
|}<br />
<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top; padding:15px;"<br />
|-<br />
|<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata ==<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead.<br />
<br />
[[Image:Ben_Cammarata.jpg|200px|thumb|left]]<br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
== Malinka Walaliyadde ==<br />
Malinka doesn’t have a nickname because there was no one around to nickname him when he invented the universe.<br />
<br />
== Kevin “Amoeba” Cheng ==<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Cheng. Look for the amoeba at the jamboree. <br />
<br />
|-<br />
<br />
|}</div>Bammarata89http://2009.igem.org/File:Ben_Cammarata.jpgFile:Ben Cammarata.jpg2009-10-22T01:45:25Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/File:Alyssa_Henning.jpgFile:Alyssa Henning.jpg2009-10-22T01:45:01Z<p>Bammarata89: </p>
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<div></div>Bammarata89http://2009.igem.org/File:Team_photo.JPGFile:Team photo.JPG2009-10-22T01:43:21Z<p>Bammarata89: </p>
<hr />
<div></div>Bammarata89http://2009.igem.org/Team:CornellTeam:Cornell2009-10-22T01:37:10Z<p>Bammarata89: /* Project Description */</p>
<hr />
<div>[[Image:CUGEM Banner.png|800px|center]]<br />
{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
<br />
=Project Abstract=<br />
Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture, Itai-Itai disease, infertility, damage to the central nervous and immune systems, cancer development and ultimately death. For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. Of late, cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems especially in developing countries where there is little regulation of industrial waste products. The goal of our project is to create a low-cost bio-sensor by using the metal ion homeostasis system of Bacillus subtilis to quantify environmental levels of cadmium in drinking water. Since B. subtilis has genetic elements that naturally respond to different concentrations of metal ions, it is an excellent system to serve as our bio-sensor chassis.</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-22T01:10:31Z<p>Bammarata89: </p>
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<div>{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
=Background=<br />
==Cadmium Contamination==<br />
<br />
''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
<br />
''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
<br />
''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
<br />
''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
<br />
==Metal Ion Homeostasis in Bacteria==<br />
<br />
''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
<br />
=Design=<br />
<br />
== Chassis ==<br />
''Bacillus Subtillis'' is a gram-positive bacteria, 5-10uM in length. The reason it was chosen to be used for this project was that it contains a complex metal-ion homeostasis system. This system was the basis of our biosensor. Also, ''B. Subtillis'' cells have a well characterized genome and are naturally competent, allowing for a relatively simple transformation method.<br />
<br />
== Proposed Design ==<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
<br />
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
<br />
'''[Briefly describe the two modules and the differences between them]'''<br />
<br />
== 1st Module ==<br />
<br />
Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
<br />
[[Image:Module 1 Inactive.GIF|600px|center]]<br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
<br />
== 2nd Module ==<br />
<br />
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
<br />
[[Image:Module 2 Inactive.GIF|600px|center]]<br />
[[Image:Module 2 Active.GIF|600px|center]]<br />
<br />
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
<br />
<br />
=== References ===<br />
<br />
T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
<br />
Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
<br />
Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
<br />
Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
<br />
Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
<br />
Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
<br />
Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-22T01:08:44Z<p>Bammarata89: /* Purpose */</p>
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<div>{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
=Background=<br />
==Cadmium Contamination==<br />
<br />
''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
<br />
''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
<br />
''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
<br />
''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
<br />
==Metal Ion Homeostasis in Bacteria==<br />
<br />
''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
<br />
=Design=<br />
<br />
== Chassis ==<br />
''Bacillus Subtillis'' is a gram-positive bacteria, 5-10uM in length. The reason it was chosen to be used for this project was that it contains a complex metal-ion homeostasis system. This system was the basis of our biosensor. Also, ''B. Subtillis'' cells have a well characterized genome and are naturally competent, allowing for a relatively simple transformation method.<br />
<br />
== Proposed Design ==<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
<br />
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
<br />
'''[Briefly describe the two modules and the differences between them]'''<br />
<br />
== 1st Module ==<br />
<br />
Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
<br />
[[Image:Module 1 Inactive.GIF|600px|center]]<br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
<br />
== 2nd Module ==<br />
<br />
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
<br />
[[Image:Module 2 Inactive.GIF|600px|center]]<br />
[[Image:Module 2 Active.GIF|600px|center]]<br />
<br />
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
<br />
=== The Experiments ===<br />
<br />
<br />
<br />
<br />
=== Part 3 ===<br />
=== References ===<br />
<br />
T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
<br />
Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
<br />
Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
<br />
Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
<br />
Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
<br />
Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
<br />
Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-22T01:07:01Z<p>Bammarata89: /* Chassis */</p>
<hr />
<div>{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
=Background=<br />
==Cadmium Contamination==<br />
<br />
''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
<br />
''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
<br />
''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
<br />
''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
<br />
==Metal Ion Homeostasis in Bacteria==<br />
<br />
''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
<br />
=Design=<br />
<br />
== Chassis ==<br />
''Bacillus Subtillis'' is a gram-positive bacteria, 5-10uM in length. The reason it was chosen to be used for this project was that it contains a complex metal-ion homeostasis system. This system was the basis of our biosensor. Also, ''B. Subtillis'' cells have a well characterized genome and are naturally competent, allowing for a relatively simple transformation method.<br />
<br />
== Purpose ==<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
<br />
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
<br />
'''[Briefly describe the two modules and the differences between them]'''<br />
<br />
== 1st Module ==<br />
<br />
Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
<br />
[[Image:Module 1 Inactive.GIF|600px|center]]<br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
<br />
== 2nd Module ==<br />
<br />
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
<br />
[[Image:Module 2 Inactive.GIF|600px|center]]<br />
[[Image:Module 2 Active.GIF|600px|center]]<br />
<br />
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
<br />
=== The Experiments ===<br />
<br />
<br />
<br />
<br />
=== Part 3 ===<br />
=== References ===<br />
<br />
T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
<br />
Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
<br />
Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
<br />
Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
<br />
Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
<br />
Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
<br />
Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-22T01:06:37Z<p>Bammarata89: /* 1st Module */</p>
<hr />
<div>{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
=Background=<br />
==Cadmium Contamination==<br />
<br />
''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
<br />
''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
<br />
''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
<br />
''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
<br />
==Metal Ion Homeostasis in Bacteria==<br />
<br />
''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
<br />
=Design=<br />
<br />
== ''Chassis'' ==<br />
''Bacillus Subtillis'' is a gram-positive bacteria, 5-10uM in length. The reason it was chosen to be used for this project was that it contains a complex metal-ion homeostasis system. This system was the basis of our biosensor. Also, ''B. Subtillis'' cells have a well characterized genome and are naturally competent, allowing for a relatively simple transformation method. <br />
<br />
== Purpose ==<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
<br />
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
<br />
'''[Briefly describe the two modules and the differences between them]'''<br />
<br />
== 1st Module ==<br />
<br />
Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
<br />
[[Image:Module 1 Inactive.GIF|600px|center]]<br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
<br />
== 2nd Module ==<br />
<br />
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
<br />
[[Image:Module 2 Inactive.GIF|600px|center]]<br />
[[Image:Module 2 Active.GIF|600px|center]]<br />
<br />
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
<br />
=== The Experiments ===<br />
<br />
<br />
<br />
<br />
=== Part 3 ===<br />
=== References ===<br />
<br />
T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
<br />
Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
<br />
Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
<br />
Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
<br />
Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
<br />
Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
<br />
Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-22T01:05:45Z<p>Bammarata89: /* Design */</p>
<hr />
<div>{|align="justify"<br />
|<br />
{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
=Background=<br />
==Cadmium Contamination==<br />
<br />
''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
<br />
''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
<br />
''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
<br />
''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
<br />
==Metal Ion Homeostasis in Bacteria==<br />
<br />
''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
<br />
=Design=<br />
<br />
== ''Chassis'' ==<br />
''Bacillus Subtillis'' is a gram-positive bacteria, 5-10uM in length. The reason it was chosen to be used for this project was that it contains a complex metal-ion homeostasis system. This system was the basis of our biosensor. Also, ''B. Subtillis'' cells have a well characterized genome and are naturally competent, allowing for a relatively simple transformation method. <br />
<br />
== Purpose ==<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
<br />
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
<br />
'''[Briefly describe the two modules and the differences between them]'''<br />
<br />
== 1st Module ==<br />
<br />
Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
<br />
[[Image:Module 1 Inactive.GIF|600px|center]]<br />
[[Image:Module 1 Active.GIF|600px|center]]<br />
<br />
== 2nd Module<br />
<br />
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
<br />
[[Image:Module 2 Inactive.GIF|600px|center]]<br />
[[Image:Module 2 Active.GIF|600px|center]]<br />
<br />
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
<br />
=== The Experiments ===<br />
<br />
<br />
<br />
<br />
=== Part 3 ===<br />
=== References ===<br />
<br />
T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
<br />
Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
<br />
Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
<br />
Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
<br />
Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
<br />
Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
<br />
Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89http://2009.igem.org/Jamboree/Schedule/Practice_sessionsJamboree/Schedule/Practice sessions2009-10-22T00:50:03Z<p>Bammarata89: /* Friday October 30 : Practice Talks sign-up sheet */</p>
<hr />
<div>== Friday October 30 : Practice Talks sign-up sheet ==<br />
<!-- <span style="color:#D22325; font-weight:bold;">As the wiki has now been frozen, please email [[User:Meagan | Meagan]] with your team name and the letter and # of the slot that you would like to reserve.</span><br>--><br />
<br />
Use this sign-up sheet to sign up for a slot on Friday night (October 30) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. <br />
<br />
There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment.<br />
<br />
Also, there will also be pre-registration available beginning at 6pm. Conference services will be on-site to pass out team registration boxes (see the [[Jamboree | Jamboree]] page). <br />
<br />
<br />
(Pizza and refreshments will be available on a first-come first-serve basis)<br />
<br />
<br />
<html><br />
<link rel="stylesheet" href="http://parts.mit.edu/igem07/index.php?title=User:Macowell/schedule.css&action=raw&ctype=text/css"><br />
<table class="calendar"><h2 class="date"><a name="Friday Practice">Friday, November 7</a></h2><br />
<thead><br />
<tr><br />
<th width="15%">Time</th><br />
<th>room 32-123</th><br />
<th>room 32-141</th><br />
<th>room 32-155</th><br />
<th>room 32-G449</th><br />
<th>room 32-D463</th><br />
<th>room 32-261*</th><br />
<th>room 32-262*</th><br />
<th>room 32-346*</th><br />
<th>room 32-397*</th><br />
<th>room 32-124</th><br />
<th>room 32-144</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>6:00p - 6:30p</th><br />
<td>Stanford</td><br />
<td>PKU_Beijing</td><br />
<td>UQ Australia</td><br />
<td>IBB_Pune</td><br />
<td>Freiburg_bioware</td><br />
<td>F1</td><br />
<td>G1</td><br />
<td>H1</td><br />
<td>I1</td><br />
<td>Aberdeen Scotland</td><br />
<td>K1</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30p - 7:00p</th><br />
<td>Warsaw</td><br />
<td>Southampton</td><br />
<td>IIT_Bombay_India</td><br />
<td>Bologna</td><br />
<td>Freiburg_software</td><br />
<td>F2</td><br />
<td>G2</td><br />
<td>H2</td><br />
<td>I2</td><br />
<td>J2</td><br />
<td>K2</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00p - 7:30p</th><br />
<td>IIT_Madras</td><br />
<td>Heidelberg</td><br />
<td>Osaka</td><br />
<td>Paris</td><br />
<td>Berkeley Software</td><br />
<td>F3</td><br />
<td>G3</td><br />
<td>H3</td><br />
<td>I3</td><br />
<td>J3</td><br />
<td>K3</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30p - 8:00p</th><br />
<td>Tokyo_Tech</td><br />
<td>Tsinghua</td><br />
<td>UNIPV-Pavia</td><br />
<td>D4</td><br />
<td>METU-gene</td><br />
<td>F4</td><br />
<td>G4</td><br />
<td>H4</td><br />
<td>I4</td><br />
<td>J4</td><br />
<td>K4</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00p - 8:30p</th><br />
<td>KULeuven</td><br />
<td>Groningen</td><br />
<td>EPF-Lausanne</td><br />
<td>D5</td><br />
<td>E5</td><br />
<td>F5</td><br />
<td>G5</td><br />
<td>H5</td><br />
<td>I5</td><br />
<td>J5</td><br />
<td>K5</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30p - 9:00p</th><br />
<td>USTC</td><br />
<td>NTU_Singapore</td><br />
<td>Todai-Tokyo</td><br />
<td>D6</td><br />
<td>E6</td><br />
<td>F6</td><br />
<td>G6</td><br />
<td>H6</td><br />
<td>I6</td><br />
<td>J6</td><br />
<td>K6</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00p - 9:30p</th><br />
<td>USTC_Software</td><br />
<td>TorontoMaRSDiscovery</td><br />
<td>UC_Davis</td><br />
<td>D7</td><br />
<td>E7</td><br />
<td>F7</td><br />
<td>G7</td><br />
<td>H7</td><br />
<td>I7</td><br />
<td>J7</td><br />
<td>K7</td><br />
</tr><br />
<tr class="even"><br />
<th>9:30p - 10:00p</th><br />
<td>British_Columbia</td><br />
<td>BIOTEC-Dresden</td><br />
<td>Chiba</td><br />
<td>Waterloo</td><br />
<td>VictoriaBC</td><br />
<td>CORNELL UNIVERSITY</td><br />
<td>G8</td><br />
<td>H8</td><br />
<td>I8</td><br />
<td>J8</td><br />
<td>K8</td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br />
<br />
'''Important information for rooms marked with an asterisk (*):'''<br />
* Your team will be contacted to coordinate having an iGEM staff member escort you to these rooms as they are in a limited-access part of the Stata Center.<br />
* These rooms are smaller conference rooms throughout the Stata Center. <br />
* Saturday sessions will not be held in these rooms but in order to accommodate all teams who would like to practice their presentations in the 4-hour period on Friday night, we must open these rooms for practice sessions.</div>Bammarata89http://2009.igem.org/Team:Cornell/TeamTeam:Cornell/Team2009-10-22T00:14:24Z<p>Bammarata89: </p>
<hr />
<div>{| style="border:1px solid #cef2e0; background:#f5fffa; color:#008811; vertical-align:top;" cellpadding="3" cellspacing="1" width="800px" align="center" font-size:"1.3em"<br />
!align="center"|[[Team:Cornell|Home]]<br />
!align="center"|[[Team:Cornell/Team|The Team]]<br />
!align="center"|[[Team:Cornell/Project|The Project]]<br />
!align="center"|[[Team:Cornell/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Cornell/Notebook|Notebook]]<br />
|}<br />
<br />
== '''Team Description''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top;"<br />
|-<br />
|The Cornell iGEM team is a multidisciplinary team with 6 undergraduates with bioengineering, material science, biology and chemistry majors. Our rookie year has been an empowering year where the traditional listeners of knowledge have become creators. <br />
|-<br />
|}<br />
<br />
== '''Team Members''' ==<br />
{|style="border:1px solid #008811; background:#ccffcc; vertical-align:top;"<br />
|-<br />
|rowspan="3"|<br />
<br />
'''Advisors:'''<br />
<br />
*''' Carl Batt''': Professor, Food Science<br />
*''' Maki Inada''': Adjunct Professor, Molecular Biology and Genetics<br />
*''' Xiling Shen ''': Assistant Professor, Electrical and Computer Engineering<br />
<br />
<br />
= Undergrads =<br />
<br />
== Xing “Xingularity” Xiong ==<br />
Born in the ghettos of the LHC (Large Hadron Collider), Xing Xiong had an aptitude for physics and hardcore rapping. He was spitting out rhymes by the age of two and three quarters, and upon reaching high school, was dubbed “Xingularity” by his peers. Xing brought his rhymes to Cornell, and during his interview for joining the Cornell GEM team, his rap performance was so phenomenal that he immediately took over as Team Leader, no questions asked.<br />
<br />
== Bernard “Big Ben” Cammarata ==<br />
Originating from the small town of Southborough, Massachusetts, Ben always had big things on his mind. Like real big. Cornell big. After being accepted into Cornell in 2007 (he is currently a junior BEE major), Ben set his sights on curing all the world’s problems. How did he plan on doing this? Well, establishing a universal dictatorship solely under his control did not work out so well, so he decided to join the Cornell Genetically Engineered Machines team instead. <br />
<br />
== Alyssa “Adrenaline” Henning ==<br />
Born in the town of Bakersfield, California, Alyssa rode her first roller coaster at the age of six months. Her second roller coaster ride is scheduled for when she gets off her first; she has been riding the same roller coaster continuously for her entire life. Somehow, she managed to ace high school, get accepted into Cornell, and join the iGEM team, all from the seat of her 3 by 3 coaster car. How does she go to the bathroom? It’s a very simple process: magic. Since she is unable to leave her coaster car, she will be sending a surrogate to the jamboree in her place. <br />
<br />
== Matthew “Ultraman” Hall ==<br />
Matthew Hall, born on the rings of Saturn, is the most interesting man in the world. At the age of three, he ran his first ultramarathon (100 miles), earning the same “Ultraman.” At the age of five, he cured world hunger, only to uncure it when someone stole is nachos. At the age of 10, he counted to infinity, twice. At the age of 18, he was accepted into one of the most prestigious schools in world. Matthew Hall’s tears can cure cancer, but sadly, he has never cried.<br />
<br />
== Malinka ==<br />
Malinka doesn’t have a nickname because there was no one around to nickname him when he invented the universe. <br />
<br />
== Kevin “Amoeba” Cheng ==<br />
Kevin reproduced asexually, creating his present day form and earning him the nickname “Amoeba.” Unfortunately, his identical copy was washed down a laboratory sink before it could fully develop, so there is only one Kevin “Amoeba” Kim. Look for the amoeba at the jamboree. <br />
<br />
<br />
<br />
|<br />
<gallery><br />
Image:Team_member_1.png|Team member 1<br />
Image:Team_member_2.png|Team member 2<br />
Image:Team_member_3.png|Team member 3<br />
Image:Team_member_4.png|Team member 4<br />
Image:Team_member_5.png|Team member 5<br />
Image:Team_member_6.png|Team member 6<br />
Image:Team_member_6.png|Team member 7<br />
</gallery><br />
|}</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-17T20:42:14Z<p>Bammarata89: /* References */</p>
<hr />
<div>== Project Details==<br />
=== Project Background ===<br />
<br />
<br />
<br />
Cadmium Contamination<br />
<br />
''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
<br />
''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
<br />
''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
<br />
''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
<br />
Metal Ion Homeostasis in Bacteria<br />
<br />
''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
<br />
=== Design ===<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
<br />
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
<br />
>Briefly describe the two modules and the differences between them<<br />
<br />
Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
<br />
[[Image:Module 1 Inactive.jpg|600px|center]]<br />
[[Image:Module 1 Active.JPG|600px|center]]<br />
<br />
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
<br />
[[Image:Module 2 Inactive.JPG|600px|center]]<br />
[[Image:Module 2 Active.jpg|600px|center]]<br />
<br />
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
<br />
=== The Experiments ===<br />
<br />
<br />
<br />
<br />
=== Part 3 ===<br />
=== References ===<br />
<br />
T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
<br />
Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
<br />
Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
<br />
Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
<br />
Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
<br />
Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
<br />
Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
<br />
Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89http://2009.igem.org/Team:Cornell/ProjectTeam:Cornell/Project2009-10-17T20:41:51Z<p>Bammarata89: /* References */</p>
<hr />
<div>== Project Details==<br />
=== Project Background ===<br />
<br />
<br />
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Cadmium Contamination<br />
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''Health Effects:'' Cadmium (Cd) is a toxic heavy metal which has no known biological function. Ingestion of Cd-contaminated water can induce diarrhea, severe vomiting, bone fracture (Itai-Itai disease), infertility, damage to the central nervous and immune systems, cancer development and ultimately death. Once affected by Cadmium, there is no effective treatment for humans (Agency for Toxic Substances and Disease Registry). For instance, in late 20th century Japan, cadmium contamination of water in the Jingzu River led to significant kidney problems and Itai-Itai bone disease in a fairly large population. In another study, Cd concentration was directly related to mortality rate1.<br />
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''Level of danger:'' Of late, Cadmium levels have been better controlled by industrial regulation, but it still poses formidable problems. The Environmental Protection Agency (EPA) has set cadmium level regulations at a maximum of 5µg/L in drinking water. Of the 1,669 hazardous waste sites on the EPA's National Priorities List (NPL), 1,014 contain Cadmium. The Australian National Pollutant Inventory (NPI) has ranked Cd number 6 out of 400 of the most toxic substances based on health and environmental hazards and human and environmental exposure to the substance. According to the NPI, Cd has a total hazard rating of 4.3 as compared to 2.8 for Carbon Monoxide and 4.0 for Arsenic.<br />
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''Extent of the problem:'' Major sources of Cd contamination include fertilizers, sewage sludge, manure and atmospheric deposition. The amount of Cd released into the environment from human activities has actually been about 10 times greater than the amount predicted from natural sources.2 Likewise, the amount of Cd released into the agricultural environment has also increased significantly over the last century.3 In fact, Cd-contaminated sewage is often used for irrigation purposes in many parts of the world, especially in developing nations.2 Crops grown in these Cd-contaminated soils are then sold in markets without any detoxification treatment. As of now, consuming Cd-contaminated crops is one of the greatest Cadmium-related problems for humans. In a study conducted in Faisalabad in Pakistan, it was found that untreated sewage used for irrigation had contamination levels that were three times the allowed level (0.03 mg.L−1 as compared to 0.01 mg.L−1) for irrigation water. Furthermore, the study found that consuming crops grown in these Cd-contaminated soils for extended periods of time can cause high levels of Cd to accumulate in humans which would lead to a number of illnesses.2 Since similar irrigation methods are used in a number of other developing countries, it would be extremely useful to have a simple method of measuring Cadmium levels in water before using it for mass irrigation purposes. <br />
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''Current Cd measurement techniques:'' Current Cd measurement techniques: Transmission-based localized surface plasmon resonance (LSPR) fiber-optic probes4, prompt gamma-ray neutron activation analysis (PGNAA) 8, and a solid surface fluorescence based flow-through optosensor for detecting cadmium in drinking water9 have been developed to determine cadmium ion concentration, but the cost efficiencies of such instruments significantly exceed the cost of a simple bacterial biosensor. <br />
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Metal Ion Homeostasis in Bacteria<br />
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''Resistance mechanisms:'' Some heavy metals, such as manganese, iron and zinc, are essential to micro-organisms as trace nutrients, in contrast to others such as cadmium and lead, which have no known beneficial roles. However, all heavy metals are toxic at high (micro- or millimolar) concentrations. Yet it is widely known that certain bacteria are capable of growing in metal contaminated areas. These bacteria are usually adapted to the presence of toxic metals by genetically encoded resistance mechanisms, whose expression is precisely regulated. Specifically, these resistance mechanisms towards many toxic metals work by excretion of the metal by an energy-dependent pump in the cell membrane.<br />
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=== Design ===<br />
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes. <br />
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Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].<br />
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>Briefly describe the two modules and the differences between them<<br />
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Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.<br />
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[[Image:Module 1 Inactive.jpg|600px|center]]<br />
[[Image:Module 1 Active.JPG|600px|center]]<br />
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Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. MntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.<br />
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[[Image:Module 2 Inactive.JPG|600px|center]]<br />
[[Image:Module 2 Active.jpg|600px|center]]<br />
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By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).<br />
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=== The Experiments ===<br />
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=== Part 3 ===<br />
=== References ===<br />
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[T. Ishihara, E. Kobayashi, Y. Okubo, Y. Suwazono, T. Kido, M. Nishijyo, H. Nakagawa and K. Nogawa (2001), Association between cadmium concentration in rice and mortality in the Jinzu river basin, Japan, Toxicology 163, p.23-28 <br />
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Qadir, A. Ghafoor and G. Murtaza (2000), Cadmium Concentration in Vegetables Grown on Urban Soils Irrigated with Untreated Municipal Sewage, Environment, Development and Sustainaility 2(1), p.13-21.<br />
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Jones, K.S., Jackson, A. and Johnston, A.E (1992), Evidence for an increase in the Cd content of herbage since the 1860s, Environ. Sci. Technol. 26, p.834-836<br />
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Lin, Tsao-Jen, and Mon-Fu Chung (2009), Detection of Cadmium by a Fiber-Optic Biosensor Based on Localized Surface Plasmon Resonance, Biosensors and Bioelectronics 24(5), p.1213-8.<br />
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Helmann, John D., Qiang Que (2000), Manganese homeostasis in Bacillus subtilus is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology 35(6), p.1454-1468 <br />
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Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40 <br />
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Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40<br />
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Grazman, B.L., and Schweikert, E.A (2005). A brief review of the determination of cadmium by prompt gamma-ray neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 152(2), p.497 – 506<br />
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Garcia-Reyes et al. Sensing of trace amounts of cadmium in drinking water using a single fluorescence-based optosensor. Microchemical Journal 82(1), p.94-99</div>Bammarata89