Team:Harvard/Daily

Daily Lab Notebook

Week 1: 6/10/09 - 6/12/09

First official meeting of the summer: finalizing the project details, setting up a tentative schedule.

We spent the first meeting narrowing in on the system we would use for our upcoming projects. After comparing the bacterial light sensing system with that of the modified yeast 2-hybrid system a consensus was reached to attempt the yeast system first. The yeast phytochrome based system seemed more sensitive and the bistable light inducible system would give us an added element of regulation, in that the system could be rapidly reset using IR wavelengths. This would be useful in a "bio-blackboard" configuration.

As a backup though, we should be prepared to revert to the bacterial system.

The potential problem for the yeast system is that phycocyanobilin (PCB) needs to be exogenously supplemented. It may need to be extracted from a suitable (algae?) source or the bacterial PCB biosyntheis pathway may need to be cloned into yeast, if possible. Also, if using yeast as a "sender" with red-luciferase, the luciferin substrate for luciferase also needs to be added externally. Uptake should occur if the media is made acidic.

David pointed out a more recent intein system that also utilizes the same Phytochrome/PIF3 domains that the modified yeast two-hybrid system uses.

We carefully studies schematics of the various light responsive genetic systems, including an attempt at adding an "inverter" component into the , from a few years ago, and the system created by .

Week 2: 6/15/09 - 6/19/09

Lab Meeting Notes.
DB3.1 LB absorbance and scattering? What did the old IGEM team dilute in? Intein people had a modification to the PCB extraction protocol—look that up. PCR the genes we need out of a cDNA library. Churchill lab has everything?—ask them for Arabadopsis cDNA library. Arabadopsis 2 hybrid library Email Woody Hastings? Fluorescent dinoflagellates! Shake them to read by.

Assembly of low-promoter from oligos. DTT is a reducing agent, ATP is energy, required for various enzynmes in the system to work.

First step is to take phosphorylate the resuspended oligos. The 5 prime ends need to be phosphorylated so that they can be ligated. Oligos normally come without the five prime phosphate. Mix equimolar amounts of the four oligos and label oligo mix, 20 uL of each oligo. Use PCR tube. 20 uL H20 6 ul oligo mix 4 ul 10x PNK buffer 4 uL DTT 4 ul ATP 2 ul PNK Total: 40 uL…….keep on ice. Samples put in thermocycler. See protocol for program, called “Assembly”.

Tuesday For obtaining the PhyA, PhyB, and PIF3 genes, we have a number of methods we are pursuing. 1. Obtain cDNAs from another lab a. Tuesday—The Mathews lab is willing to give us PhyA, but does not have PhyB or PIF3. They can help us with the construction of the PhyB and PIF3. We should ask them if they have a protocol they follow…. b. Wednesday--I have emailed the Mathews lab back, and will hopefully be meeting with Sarah Mathews later this week. 2. Obtain cDNAs from Biobricks registry a. Tuesday--Pulled PhyB and PIF3 from the registry, as well as the PCB synthesis genes. The PCB synthesis genes look good according to registry sequence, but PhyB and PIF3 cite sequencing irregularities, so I am not hopeful. b. Wednesday—Oliver compared the sequences from NCBI to the sequences given in the BioBricks registry for the PhyB and PIF3 and it does not look like they are correct, so that is not promising. We will continue with growing up the PCB biosynthetic enzymes though. 3. Obtain cDNAs from a cDNA library a. Tuesday—No one seems to have a sample of the cDNA library we can have. Asked in the lab plant kid from 261r was in, he’s checking for us but is not hopeful. Purchase of a cDNA library is too expensive, $1200) 4. Obtain Arabidopsis plant and make a cDNA library, and from that clone out the genes of interest a. Tuesday—The Pierce lab has kindly given us two lovely Arabidopsis plants, and we have ordered the necessary kits and mortar and pestle to do the RNA extraction. Tomorrow must design primers for sequencing and for cloning the genes out…these can be multipurpose. b. Wednesday-- 5. Obtain a library from TAIR. Arabidopsis.org a. Wednesday—We are going to order libraries from the Arabidopsis people. They are very cheap, $5 a line, with $125 shipping fee total per order, so it’s fairly cheap. Shipping is combined for all orders. b. 4 different libraries ordered.

Initial email to Mathews Hi Dr. Mathews,

My name is Amrita Goyal, and I am a member of the Harvard iGEM team. We are looking for the cDNA for the PhyB, PhyA, or PIF3 components of the light-activated signalling system in plants. We were wondering if you had these genes available in a suitable vector that we could obtain. Our goal is to subclone these genes into a yeast-two-hybrid system. We would really appreciate it if you had a sample available that one of our teammates could pick up. Also, if you have an extraction protocol for phycocyanobilin, or know someone who does, we would also appreciate that information. Thank you so much! Sincerely, Amrita Goyal and the Harvard iGEM team

Mathew’s Response to me Hi Amrita, Sorry for the delay in responding -- I've been doing jury duty. We can help with a PHYA cDNA clone, in either pUC 18 or a bluescript vector. We don't have PHYB or PIF clones, or an Arabidopsis cDNA library. But depending on your timeframe, I could help with PHYB and PIF clones. For a phycocyanobilin extraction protocol, I recommend you get in touch with someone in Clark Lagarias' lab (cc'd on this message). Their web page is at: http://www.mcb.ucdavis.edu/faculty-labs/lagarias/ We may have some phytochromobilin, but we have no phycocyanobilin. Let me know when you'd like to pick up some PHYA clone, and I'd love to hear a bit more about your experiments. Cheers, Sarah

My response to Mathews Hi Professor Mathews, I hope that jury duty went well! If we could get the PHYA cDNA in the pUC18 vector from you that would be fantastic. We would very much appreciate your help with making the PHYB and PIF3. We acquired a couple of Arabidopsis plants from a grad student in Naomi Pierce's lab yesterday and we were planning to do an RNA extraction and clone the PHYB and PIF3 out ourselves, if you think that is the best way to proceed. If you think the phycochromobilin would work, we would also appreciate a sample of that. We did get Spirulina and are planning to attempt a PCB extraction soon. What we are planning to do for our iGEM project is to create a cellular blackboard, using the Phy system in yeast. We want to use the PhyA and/or PhyB to turn on transcription of a green luciferase, so we can "write" on the blackboard using a red laser pointer. We would then "erase" the blackboard with far-red light to turn off luciferase transcription (although we will just have to wait for the luciferase to degrade then). We were also thinking of engineering a system where we have two populations of yeast cells communicate with eachother using light. We would turn on production of a red luciferase in one population by shining a laser on it. The light produced by that population would then be used to turn on production of the luciferase in another population of cells. If you have any thoughts or advice on either of these projects please let me know! I can come by any time today or after lunch tomorrow, let me know when is most convenient for you! I actually have a couple of questions for you if you have a few minutes to talk in person in the next couple of days. Let me know what your schedule looks like! Thanks, Amrita

Response from Lagarias, jclagarias@ucdavis.edu Colleagues, I have been contacted for a related request by a Wash University iGEM Team. I told them that we would have to complete an MTA to send any plasmids (and we do not have PIF clones that we are at liberty to send; they were obtained from other labs). FYI, I have attached a protocol for PCB isolation. Best. Clark Lagarias

PREPARATION OF PHYCOCYANOBILIN Lagarias Lab Method 3E-phycocyanobilin isolation: Lagarias Lab Method (Terry, MJ, MD Maines, and JC Lagarias. 1993. Inactivation of Phytochrome-Chromophore and Phycobiliprotein-Chromophore Precursors by Rat Liver Biliverdin Reductase. J. Biol. Chem. 268(35):26099-26106): 3E-phycocyanobilin (PCB) was prepared from lyophilized Spirulina platensis (Sigma) using a method similar to that described by Beale and Cornejo (Beale & Cornejo (1991a) J. Biol. Chem. 266, 22328-22332; Beale & Cornejo (1991b) J. Biol. Chem. 266, 22333-22345). 1. Spirulina powder was rehydrated in deionized water (30 ml/g dry weight) for 10 min. 2. The slurry was centrifuged at 30,000 x g for 20 min and the deep blue supernatant was decanted. 3. Phycocyanin was precipitated from this supernatant with 1% (w/v) TCA by incubation for 1 h at 4oC in the dark and then collected by centrifugation at 30,000 x g for 20 min. 4. After washing with methanol (2 x 20 ml/g Spirulina powder), the blue pellet was resuspended in methanol (2 ml/g Spirulina powder) containing 1 mg/ml HgCl2. 5. Following incubation for 20 h at 42oC in darkness, the protein was removed by centrifugation at 10,000 x g for 10 min. 6. 2-Mercaptoethanol (1 ul/ml) was then added to precipitate the dissolved mercuric ion, which was also removed by centrifugation (30,000 x g for 10 min). 7. The crude bilin mixture was then diluted 10-fold with 0.1% (v/v) trifluoroacetic acid and applied to a C18 Sep-Pak cartridge (Waters-Millipore Corp., Milford, MA). The Sep-Pak cartridge was sequentially washed with 0.1% (v/v) trifluoroacetic acid (2 x 3 ml) and acetonitrile/0.1% trifluoroacetic acid (20:80; 2 x 2 ml) followed by elution of the crude bilin mixture with 3 ml acetonitrile/0.1% trifluoroacetic acid (60:40) and drying in vacuo. 8. Spectral assay of the crude bilin mixture is performed at this point (use a 1/100 dilution) and a typical yield of 4 umol phycocyanobilin per 6 g dry weight Spirulina can be obtained. 9. This crude phycocyanobilin is quite pure but can be further purified by C18 reverse phase HPLC using a Varian 5000 liquid chromatograph and a Beckman Ultrasphere ODS column (4.6 x 150 mm; 5 um particle diameter). The solvent system used was ethanol/acetone/water/acetic acid, 19:14:66:1 (v/v/v/v) with a flow rate of 1.5 ml/min, and the column eluate was monitored at 370 nm (Cornejo et al, 1992). 10. HPLC-purified 3E-phycocyanobilin was concentrated using a C18 Sep-Pak as described above, dried in vacuo and stored at -20oC. Before use, phycocyanobilin was dissolved in dimethyl sulphoxide to a concentration of 1-1.5 mM. 11. An aliquot of each stock solution was diluted 200-fold into 2% HCl/methanol to estimate the bilin concentration spectrophotometrically. The molar absorption coefficient of 47,900 M-1 cm-1 at 374 nm for 3E-phycocyanobilin (Cole, WJ, DJ Chapman, and HW Siegelman. 1967. The structure of phycocyanobilin. J. Am. Chem. Soc. 89:3643-3645) were used for these determinations.

1. HO1 a. BB—726 bp, 4425 bp. 2. PycA a. BB—750 bp, 4425 bp. 3. PhyB a. BB—1863 bp b. TAIR—3996 bp 4. PIF3 a. BB—302 bp b. TAIR—1790 bp

Truncated is better There is a truncated PhyA PhyB is main mediator of low fluence red light, conversion to Far form. W PhyA, main mediator of responses to far red light, in shaded environments. 2 modes—responses to prolonged exposure to far red light, and low fluence exposure if PhyA totally held in the dark. Still converts w red. Low fluence response does not matter on quality of light—any small exposure to visible light.

PhyA will bind PIF3, but in plantae, never. Getting phyA into nucleus FI1 needed for translocation to nucleus. Nuclear localization signal.

Lagarias—has been trying to get both phytochromobilin and the phytochrome to express concurrently in yeast, most people use the bacterial for heterologous expression. Clarke would be the person to ask to see hwat he has gotten to work. –email him he will respond.

They have primers for all Phy B and could design primers for just truncated section. Try just amplifying from genomic DNA in a single exon. Def try from genomic DNA.

Probbablyjust want to work with PhyB. In some ways could be advantageous s to work with Phy A—differs in how sensitive it is to light—once it sees light it degrades rapidly. Goes into proteosome degradation, and maybe a proteosome box is exposed? Maybe that’s just specific in Arabidopsis. It’s an E3 ligase. We need to look at specific structure of the protein. In plants PhyA builds to high levels in the dark and disappears in light—degradation and turning down. In yeast dunno how it would behave. –maybe working with a couple or three just to make sure one is robust would be good.

Def worth trying both PhyA and PhyB—espworking with full length. Don’t know if truncated has same sensitivity characteristics. PhyA is most sensitive.

You need to express the protein in the dark. The trick is the conditions under which you express the protein—use minimum minimum green light. Just put in green filters, 25 w bulb. If you can do that….The person collecting the data has a spectroradiometer and he had built a red and far-red filter to push them between the red and far red films in the cuvettes. She will see if they still have that instrument. The trick will be the conditions.

Mathews: Primer sequences to help get useful fragments—send messages to truncated clone people

You will never convert everything back from Pfr. 3 fctors effecting rate—it will dark revertat a specific rate, you can push it back with far red, and then temperature—revert faster if pulsed with far red.

We need the vectors for the yeast-two hybrid system, and the strains of yeast they used. We are particularly looking for the strains that have the UAS driven reporter if you have them.

Yeast two hybrid system Strain missing Gal4 and Gal80. Gal4 has DNA binding domain and activation domain. Binds to several promoters, one is pGAL1. There is also a GAL2 and GAL4. The strain usually has these two deletions, and those are redulated under galactorse, truned on by it. In the absence of galalctose it is blocked by Gal80. Usually in one of the GAL promoters you have GAL4 deleted and GAL80 deleted. You usually have a reporter, and you can measure by adding OMPG or whatever substrate. The strains come in two mating types, A or alpha. They are haplopd, when opposite mating types meet they fuse.

PRS303—0 means it integrates. You put in your gene of interest and you cut this plasmid in the selectable marker and linearize the plasmid and it integrates into the yeast genome and there is enough homology so youhave a good copy of the leucine gene. Andyou can have it driven under whatever promoter.

PIF3—FL PIF3—Partial PhyB—Partial

We need full length Gal4—this will

PCB questions-- Do we need to HPLC it? Will it work without it? Can we just insert the genes into the yeast and have it express the enzyme?

Resources www.addgene.org www.atcc.org www.sgd.org www.arabidopsis.org

Week 3: 6/22/09 - 6/26/09

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Week 4: 6/29/09 - 7/3/09

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Week 5: 7/6/09 - 7/10/09

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Week 6: 7/13/09 - 7/17/09

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Week 7: 7/20/09 - 7/24/09

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Week 8: 7/27/09 - 7/31/09

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Week 9: 8/3/09 - 8/7/09

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Week 10: 8/10/09 - 8/14/09

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Team

Projects

• Bacteria-to-Yeast Communication
• Characterization
• PCB Biosynthesis in Yeast
• Split Luciferase
• Yeast Cellular Blackboard
• Yeast Red Light District

Parts

References

Lab Notebook

• Daily
• Protocols
• Recipes

Ethics