Team:TorontoMaRSDiscovery/Notebook

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{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]
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!align="center"|[[Team:TorontoMaRSDiscovery/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]
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!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]
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!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modelling]]
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!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]
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!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]
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<br>
<br>
-
=April 27, 2009=
+
==Monthly Notebook==
-
#Received from Rosa (SPiT):
+
<ul>
-
#*TM0785
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/April April]
-
#**Plasmid containing encapsulin
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/May May]
-
#**Recommend transfect into bacteria and re-sequence
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/June June]
-
#**See email note regarding sequence error
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/July July]
-
#*0.5 microliters TMG DNA 100 microgram/microliter
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/Augst August]
-
#**Use 0.4 microliter for 50 microliter PCR reaction =August 1, 2009=
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/September September]
-
#Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
+
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/October October]
-
#The rest of the encapsulin cultures were stocked with 20% glycerol
+
</ul>
-
#6 digestions were done: Enc1, Enc6, Enc -ve, BB5, BB7, and C
+
-
#*16ul of BB5 plasmid was used
+
-
#*500ng of plasmid were used for the others
+
-
#The digestions were run on a 1.3% agarose gel in TAE (from here on, unless otherwise specified, all gels were 1.3% agarose)
+
-
#*BB5 was confirmed and all other parts were correct as well
+
-
#Overnight ligation of 7+Enc in the PCR machine
+
-
=August 2, 2009=
+
==Summary of Results==
-
#Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
+
-
#Digested these plasmid samples and ran on gel with negative control (straight from fridge)
+
-
#*The 3kbp in the digest of 1+2 Sample 5 was not expected
+
-
#*Comparing the 2 samples of 3+2, the digest of sample 1 did not have a 700bp band, and the undigested sample 1 was about 500bp shorter than the undigested sample 2. We suspect the plasmid in sample 1 did not take up the 3+2 part and somehow recircularized.
+
-
#Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates
+
-
=August 3, 2009=
+
{| border="1" cellpadding="5" cellspacing="0"
-
#No colonies were found on 7+Enc plates
+
|'''Part Type'''
-
 
+
|'''Arbitrary Name'''
-
=August 4, 2009=
+
|'''Registry Code'''
-
#Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
+
|'''Construct Used For'''
-
#Digested BB4, BB5 and C plasmid, and ran them on a gel
+
|'''Status'''
-
#*All bands were of expected sizes.
+
|'''Transformants Stocked?'''
-
# Started overnight ligation of 4+5 into C plasmid
+
|'''Antibiotic Resistant, Backbone Plasmid Visualized?'''
-
 
+
|'''Part Visualized?'''
-
=August 5, 2009=
+
|'''Sequenced?'''
-
#Transfected overnight ligation and plated them on C plates
+
|----
-
#Plated new C plates (probably meant poured)
+
|rowspan="2"|
-
#Miniprepped overnight cultures of BB1+2 and K plasmid
+
'''New Parts'''
-
 
+
|Encapsulin (Enc)
-
=August 6, 2009=
+
|K192000
-
#Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
+
|Encapsulin
-
#The digests along with negative controls were ran on a gel
+
|Confirmed
-
#*The 1+2 insert was not seen on the gel, probably because it is too small
+
|Yes
-
#*The ccdb gene (~600bp) was not seen on the gel
+
|Yes
-
 
+
|Yes
-
=August 7, 2009=
+
|Yes
-
#Started overnight cultures of 1+2 sample 1,2,4
+
|----
-
#Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
+
|eCFPtgt
-
#Plated DH5alpha cells as a control on plain LB plates
+
|K192001
-
#**This is thumotoga maritime genomic DNA for purpose of re-cloning <Br />
+
|CFP target protein
-
#Microcentrifuge tubes 1 and 2 placed in -20 freezer
+
|Synthesized by Mr Gene
-
 
+
|No
-
=May 15, 2009=
+
|No
-
#pH buffers received from VWR Mississauga
+
|No
-
#*pH 4 buffer (red) 500 ml
+
|Yes
-
#*pH 7 buffer (yellow) 500 ml
+
|----
-
#*pH 10 buffer (blue) 500 ml<Br />''Above are used for pH/mV Meter calibration''
+
|rowspan="13"|
-
#pH/mV meter calibrated according to manual – recorded in index
+
'''Registry Parts'''
-
#Ethanol solution (70%) made from 85% ethanol
+
|1
-
 
+
|JJ23100
-
=May 19, 2009=
+
|Control, Encapsulin
-
#2L of TE buffer made (10X TE)
+
|Confirmed
-
#:''Recipe for 2L from stock solution (10X TE)''
+
|Yes
-
#::a) 10 ml 1M Tris-HCl pH 7.5-80.0 x 2 = 20 ml
+
|Yes
-
#::b) 2 ml 0.5 M EDTA pH 8.0 x 2 = 4 ml
+
|Too small
-
#::c) 988 ml ddH20 x 2 = 1976 ml
+
|No
-
#::''To make Tris-HCl, Tris base is used and adjusted to desired pH using HCl'' <Br />
+
|----
-
#::''1 M Tris base is required of which 20 ml is required – thereby 2.4228 g Tris base (due to scale limitations, 2.42g of Tris Base was used''
+
|2
-
#500 ml of 1 M Tris Base made
+
|B0034
-
#:mass of Tris used = 1M/1000 ml x 500 ml x 121.14 g/mol = 60.57 g
+
|Control, Encapsulin
-
#:volume of water used = 500 ml
+
|Confirmed
-
#250 ml of 0.5 M EDTA solution was made
+
|Yes
-
#:mass of EDTA used = 36.53 g
+
|Yes
-
#:observations: EDTA did not dissolve in ddH2O on heat and being stirred
+
|Too small
-
 
+
|No
-
=May 21, 2009=
+
|----
-
#Retrieved autoclaved ddH20, glycerol solution
+
|3
-
#Gel Electrophoresis (test run)
+
|C0040
-
#*1% gel: 0.5 g agar mixed with 50 ml of 1X TBE
+
|Control
-
#*10X TBE diluted with 5 ml TBE + 45 ml ddH20 – did not use all 50 ml for 1 gel (gel will be too thick – make 2 instead)
+
|Confirmed
-
#*''Loading Dye'': add glycerol solution to 25 mg bromophenol blue then add dH20 to 10 ml (to make 6X)
+
|Yes
-
#*Running gel: match wells to black side, run at 120 mA
+
|Yes
-
#Visualize Gel in UV
+
|Yes
-
##Turn power on
+
|No
-
##Gel in machine face up
+
|----
-
##Close door securely
+
|4
-
##Turn white light on
+
|C0012
-
##Adjust zoom, contrast, focus from black dial on top of machine
+
|Control
-
##Turn white light off (turns on UV)
+
|Confirmed
-
##Press ‘live’ toggle – acq. Should be 0.4 sec.
+
|Yes
-
##Print if desired or save on floppy disk
+
|Yes
-
##Turn power off
+
|Yes
-
##Dispose of gel in proper container
+
|No
-
##Close door
+
|----
-
 
+
|5
-
=May 25, 2009=
+
|B0015
-
#Made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)
+
|Control, Encapsulin
-
 
+
|Confirmed
-
=May 26, 2009=
+
|Yes
-
#Took overnight cultures from incubator
+
|Yes
-
#Inoculated 2 500 ml flasks with 25 ml of overnight culture (1 flask/culture)
+
|Yes
-
#Placed 500 ml flasks into incubator at 37 degrees Celcius
+
|No
-
#Grew overnights of DB3.1 from Waterloo (thanks :))
+
|----
-
 
+
|C (Amp + C)
-
=June 3, 2009=
+
|pSB1AC3
-
#Plasmid transformed = pSB1AC3 (TEST)
+
|Assembly
-
#*Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.
+
|Confirmed
-
 
+
|Yes
-
=June 5, 2009=
+
|Yes
-
#Tet plates made
+
|Yes
-
#:Recipe for 200 ml (approx. 10 plates):
+
|No
-
#::2.2 g agar in 200 ml fresh LB
+
|----
-
#::Note: do not re-autoclave LB, it will caramelize!
+
|C (C only)
-
#:Recipe for 200 ml LB:
+
|pSB1C3
-
#::a) 1 g yeast extract
+
|Assembly
-
#::b) 2 g peptotryptone
+
|Confirmed
-
#::c) 2 g NaCl
+
|Yes
-
#::d) 200 ml water
+
|Yes
-
#Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
+
|Yes
-
#Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
+
|No
-
#Swirl and poured into prepared, labeled plates
+
|----
-
#*Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
+
|K (ccdb)
-
#Inverted and put in 37 degree incubator to dry
+
|pSB1AK3
-
 
+
|Assembly
-
=June 8, 2009=
+
|Confirmed
-
#Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)  
+
|Yes
-
#Bacterial liquid culture placed in shaker at 10:51 a.m. 
+
|Yes
-
 
+
|Yes
-
=June 9, 2009=
+
|No
-
#Digested miniprepped gel with EcoRI and SpeI
+
|----
-
#Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
+
|K (RFP)
-
#DNA Ladder made - 6 microlitres of stock used
+
|pSB1K3
-
 
+
|Assembly
-
=June 10, 2009=
+
|Confirmed
-
#Poured 10 Tet plates following procedure on June 5, 2009
+
|Yes
-
#Due to problems with DNA ladder and restriction enzyme digest yesterday, procedures were followed again today with the following adjustments
+
|Yes
-
#*DNA was diluted and run on lanes 1-5 of gel:
+
|Yes
-
#**Lane 1 - 1X
+
|No
-
#**Lane 2 - 1/6X
+
|----
-
#**Lane 3 - 1/36X
+
|Tet (ccdb)
-
#**Lane 4 - 1/10X
+
|pSB1AT3
-
#**Lane 5 - 1/100X
+
|Assembly
-
#*Restriction enzyme digest was repeated with an incubation time of 4 h at 37 degrees Celsius and heat shock at 80 degrees Celsius was done in hot water bath
+
|Confirmed
-
#*Results: Dilution did not work well (ladder could not be visualized). Digest appeared to work as 3 bands could be seen in last 2 lanes
+
|Yes
-
#*Adjustments for tomorrow:
+
|Yes
-
#**Spin down enzymes before using
+
|Yes
-
#**Overnight digest
+
|No
-
 
+
|----
-
=June 11, 2009=
+
|Tet (RFP)
-
*We did not have enough isolated plasmid DNA to justify doing another gel so we decided to perform another gel so we decided to perform another miniprep to get some more
+
|pSB1T3
-
*Used DB3.1 transformed bacteria from shaker(measured spec 0.794)
+
|Assembly
-
*Miniprep
+
|Confirmed
-
**potential issue our microcentrifuge only spins @ 10,000 not required 12,000
+
|Yes
-
*Binding DNA
+
|Yes
-
**did both optional steps preheated TE + washed w/ [[W10]]
+
|Yes
-
 
+
|No
-
*measured UV absorbance = 4.2ng/ul
+
|----
-
*in all there is ~ 74 ul of TE should indicated ~370 ng of plasmid.
+
|7
-
*Digest
+
|J13002
-
** NOTE: plasmid length w/e biobric is 3189 bp biobrick length is 3255bp this can explain lack of 2 clear bands on gel after ligation.
+
|Encapsulin
-
**nevertheless, we will perform another Gel to make sure we can properly run ladder to determine fragment sizes
+
|Transfected
-
 
+
|Yes
-
*digest measurements: 250ng plasmid all else half except BSA
+
|No
-
 
+
|No
-
=June 12, 2009=
+
|No
-
* Ran Gel
+
|----
-
** Ladder and other bands were able to be visualized however were still a little wonky
+
|Amp
-
**Suggests for improvement
+
|pSB1A3
-
***increase the glycerol [] in loading dye as there have been problems with diffusion when loading samples as the solution is not falling into the wells
+
|Assembly
-
 
+
|Transfected
-
=June 15, 2009=
+
|Yes
-
*made 1ml and 200ul aliquotes of Kanamycin 10mg/ml stock and stored at -20C
+
|No
-
**Recipe: 250ug Kanamycin + 25ml sterile water
+
|No
-
**USE : 2x200ul for final working concentration of 20ug/ml
+
|No
-
Trouble shooting ladder
+
|-
-
*it was noted that the 1x TBE buffer recipe was off. The
+
|rowspan="7"|
-
 
+
'''Assembled Parts'''
-
=August 1, 2009=
+
|----
-
#Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
+
|Enc in C (Amp+C)
-
#The rest of the encapsulin cultures were stocked with 20% glycerol
+
|
-
#6 digestions were done: Enc1, Enc6, Enc -ve, BB5, BB7, and C
+
|Submission, Encapsulin
-
#*16ul of BB5 plasmid was used
+
|Confirmed
-
#*500ng of plasmid were used for the others
+
|Yes
-
#The digestions were run on a 1.3% agarose gel in TAE
+
|Yes
-
#*BB5 was confirmed and all other parts were correct as well
+
|Yes
-
#Overnight ligation of 7+Enc in the PCR machine
+
|Yes
-
 
+
|----
-
=August 2, 2009=
+
|1+2 in C (Amp+C)
-
#Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
+
|
-
#Digested these plasmid samples and ran on gel with negative control (straight from fridge)
+
|Control, Encapsulin
-
#*The 3kbp in the digest of 1+2 Sample 5 was not expected
+
|Confirmed
-
#*Comparing the 2 samples of 3+2, the digest of sample 1 did not have a 700bp band, and the undigested sample 1 was about 500bp shorter than the undigested sample 2. We suspect the plasmid in sample 1 did not take up the 3+2 part and somehow recircularized.
+
|Yes
-
#Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates
+
|Yes
-
 
+
|Too small
-
=August 3, 2009=
+
|No
-
#No colonies were found on 7+Enc plates
+
|----
-
 
+
|3+2 in C (Amp+C)
-
=August 4, 2009=
+
|
-
#Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
+
|Control
-
#Digested BB4, BB5 and C plasmid, and ran them on a gel
+
|Confirmed
-
#*All bands were of expected sizes.
+
|Yes
-
# Started overnight ligation of 4+5 into C plasmid
+
|Yes
-
 
+
|Yes
-
=August 5, 2009=
+
|No
-
#Transfected overnight ligation and plated them on C plates
+
|----
-
#Plated new C plates (probably meant poured)
+
|4+5 in Tet (ccdb)
-
#Miniprepped overnight cultures of BB1+2 and K plasmid
+
|
-
 
+
|Control
-
=August 6, 2009=
+
|Confirmed
-
#Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
+
|Yes
-
#The digests along with negative controls were ran on a gel
+
|Yes
-
#*The 1+2 insert was not seen on the gel, probably because it is too small
+
|Yes
-
#*The ccdb gene (~600bp) was not seen on the gel
+
|No
-
 
+
|----
-
=August 7, 2009=
+
|1+2+3+2 in K (RFP)
-
#Started overnight cultures of 1+2 sample 1,2,4
+
|
-
#Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
+
|Control
-
#Plated DH5alpha cells as a control on plain LB plates
+
|Transfected
-
 
+
|Yes
-
=August 8, 2009=
+
|No
-
#BB1 transformants showed many colonies
+
|No
-
#*plates (a plate full of colonies)
+
|No
-
#*-> increasing heat shock to 90s increased efficiency (Note: 3x DNA used in this transformation)
+
|----
-
#*Calvin mentioned he does heat shock for 120s
+
|1+2+Enc in K
-
#*Calvin also suggested to increase tranformation time, ie. after adding DNA, hold on ice for an hour
+
|
-
#DH-5alpha replates all grew on the plates, demonstrating LB can be re-autoclaved and poured as plates
+
|Encapsulin
-
#*there was no difference between transformants directly plated after adding LB+glucose or incubating for an hour
+
|Confirmed
-
#Miniprepped overnights (1+2) samle 1,2,4, and K
+
|Yes
-
 
+
|Yes
-
=August 10, 2009=
+
|Yes
-
#Digested plasmid samples miniprepped on Sat (1,2,4,K) and Enc, C plasmid with EcoR1 and Pst1
+
|In Progress
-
#Ran a gel for the digests
+
|----
-
#*(1+2) Sample 1 appeared to have a ~100bp band
+
|}
-
#*K plasmid digest did not show the ccdb gene (~700bp) - will start another culture with antibiotics
+
-
#Started overnight cultures of (1+2) Sample 1
+
-
 
+
-
=August 11, 2009=
+
-
#Digested 4, 5, Enc, C(E+P), C(X+S)
+
-
#*BB4 did not digest
+
-
#*5, C is stored in fridge
+
-
#Calvin suggested increasing the concentration of enzyme and increase time of digestion due to thawing enzymes over weekend in fridge
+
-
#Miniprepped 1+2(1) with new kit, got great yield ~100ng/ul
+
-
#Gel extracted C (X+S) after it was CIPed
+
-
#Overnight ligation of Enc+C in PCR machine
+
-
#Calvin gave us 2 vials of RbCl cells in -80C
+
-
#*one time use only
+
-
#*thaw and add DNA
+
-
 
+
-
=August 12, 2009=
+
-
#Transformed Enc+C ligations
+
-
#Digested BB4 again
+
-
#Started overnight ligation of 4+5
+
-
#started overnight culture of BB7
+
-
#Problem was found in C plates: too soft
+
-
#*not enough agar?
+
-
 
+
-
=August 14, 2009=
+
-
#
+

Latest revision as of 03:05, 22 October 2009

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Home The Team The Project BioBricks Modelling Bioinformatics Safety Notebook


Monthly Notebook

Summary of Results

Part Type Arbitrary Name Registry Code Construct Used For Status Transformants Stocked? Antibiotic Resistant, Backbone Plasmid Visualized? Part Visualized? Sequenced?

New Parts

Encapsulin (Enc) K192000 Encapsulin Confirmed Yes Yes Yes Yes
eCFPtgt K192001 CFP target protein Synthesized by Mr Gene No No No Yes

Registry Parts

1 JJ23100 Control, Encapsulin Confirmed Yes Yes Too small No
2 B0034 Control, Encapsulin Confirmed Yes Yes Too small No
3 C0040 Control Confirmed Yes Yes Yes No
4 C0012 Control Confirmed Yes Yes Yes No
5 B0015 Control, Encapsulin Confirmed Yes Yes Yes No
C (Amp + C) pSB1AC3 Assembly Confirmed Yes Yes Yes No
C (C only) pSB1C3 Assembly Confirmed Yes Yes Yes No
K (ccdb) pSB1AK3 Assembly Confirmed Yes Yes Yes No
K (RFP) pSB1K3 Assembly Confirmed Yes Yes Yes No
Tet (ccdb) pSB1AT3 Assembly Confirmed Yes Yes Yes No
Tet (RFP) pSB1T3 Assembly Confirmed Yes Yes Yes No
7 J13002 Encapsulin Transfected Yes No No No
Amp pSB1A3 Assembly Transfected Yes No No No

Assembled Parts

Enc in C (Amp+C) Submission, Encapsulin Confirmed Yes Yes Yes Yes
1+2 in C (Amp+C) Control, Encapsulin Confirmed Yes Yes Too small No
3+2 in C (Amp+C) Control Confirmed Yes Yes Yes No
4+5 in Tet (ccdb) Control Confirmed Yes Yes Yes No
1+2+3+2 in K (RFP) Control Transfected Yes No No No
1+2+Enc in K Encapsulin Confirmed Yes Yes Yes In Progress
Retrieved from "http://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook"