Lab Notebook

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May 15: Transformed Top10F' bacteria with the pGFP vector containing the Luciferase gene (hopefully the ligation worked this time).17:22, 15 May 2006 (EDT)

May 16: Transformation of Top10F did not work. The cause was an error in the extraction of the DNA from the the agarose gel. Completed a new agarose gel extraction of pGFP plasmid DNA, as cut by EcoRI and HindIII. Prepared a new seed of Top10F bacteria transformed with luciferase. Prepared a midiprep seed of EcoMscL.

May 17: The control of the miniprep? of luciferase was murky. This could have been due to contaminants in the LB, water, ampicillin, or pipet tips. A miniprep of both luciferase and EcoMscL was conducted. A new seed of luciferase was prepared. The miniprep of luciferase was digested with EcoRI and HindIII. The miniprep of EcoMscL was digested with XmnI. A gel with the digests was run but the EcoMscL showed no bands and the luciferase only had faint bands.

May 18: The controls of the seed of luciferase were clear. A miniprep of the new luciferase seed and the old EcoMscL seed was conducted. The same restriction enzymes were used to digest the plasmids. A gel was run with the digests. There were two faint bands on the gel with the EcoMscL when there should have been three. Thus a new batch of Stable 3 bacteria were transformed with EcoMscL through heat shock and plated. The gel with the psp-Luciferase turned out well and the gene was extracted from the gel. Ligation was not completed since the microcentrifuge tube with the pGFP cut vector could not be found due to labelling issues.

May 19: The Stable3 plates were good. We Seeded with 5 microliters of Amp in 2.5mL of LB and 2.5 mL of sterile water and placed in shaker-incubator overnight. We also ligated pGFP & Luciferase (+2 controls, c.f. lab manual) incubating at room temp for 1h. The ligation was then put in the freezer.

May 21: Put up an example of cloning for oscillator.

May 23: A transformation of the luc pGFP ligation product was performed. It was reasoned not to do transfomation on the control (with no ligase) in order to save Top Ten colonies. Also only the transformation of only the ligation with 12 microliters luciferase and 3 microliters pGFP was done, not the 6 microliter luciferase one for the same reason as mentioned above. Only half (10 microliters) of the 12 uL-ligation was used, the rest stored in freezer. The result was plated. Regarding the MSCl, the seeding from the 19th did not work, both the controls were found to be murky (ampicillin and LB as well as LB only) therefore this was performed again.

May 24: 4 or 5 colonies on plates. Seeded 3 colonies in 3 tubes with 5mL LB. [Initially believed there were no colonies and ligation didn't work. We ran a gel of the ligation to see if it contained the Luciferase gene or any DNA at all (since the extraction procedure might not have worked).] It turned out that the Gel was actually fine. There was DNA in the Gel that corresponded to the luciferase insert and the pGFP plasmid. The recombinant plasmid was not seen, but it was not necessary to see it because usually only a small amount of recombination occus. So the plates were re-examined and it was found that on the 150ul plate there were indeed some faint colonies. These colonies were then seeded, 3 colonies and 2 controls picked. Miniprep of Stable 3 bacteria with pB10 vector containing MscL was performed.

May 25: The seeding did not work. It was done again, leaving the pipet tip in the tube. The miniprep product (pB10-MscL) was digested with Xmn1 and gel was run, to check the identity of the DNA. 3 new ligations of pGFP-Luciferase were performed: 1uL-vector/15uL-insert ; 1,32/4,43 ; 4/12. -15:00, 25 May 2006 (EDT)The Gel Showed absolutely nothing, the laddder was smeared and there was no DNA in the lane.

May 26: It was found that there was nothing in the seeding, as well the new ligations/transformations did not work. Worked on midi-prep of MSCL and digested luciferase and GFP with same enzymes: EcoRI and Hind III. Ran digests on a gel and got two very faint bands in wells 5, 6 and 7 for luciferase plasmid and gene but no bands in wells 2, 3 and 4 for GFP. Cut out luciferase gene and put gel in two tubes in freezer labeled "luc insert" with the date. WE think the reason there was no GFP is because we may have cut the insert instead of the plasmid because we used GFP DNA from May 16th, DNA which could not be identified as to whether it was the whole plasmid or just the GFP gene. Decided to make a square table to increase organization and to avoid confusion. Adopted new labeling technique where paper boxes correspond to actual boxes in freezer. Everytime we put something in the freezer, we indicate on paper in the appropriate box what we put there (name of plasmid), when (date) and who did it (first name). Now a message from Horia about tomorrow's fundraising party: Hey guys I just want to say that I won't be able to come in today, I still have to run some errands for the party tomorrow. Ashwin, can you bring my notebook tomorrow? And Ashwini, are you still interested in crafting a donation box? And who wanted to make snacks/brownies/cookies again? Just let me know by email or phone, so i get this organized - in my head at least:) Horia 12:07, 26 May 2006 (EDT)

May 29:Goals for this week: produce and screen midipreps of pGFP and EcoMscL. Grow a batch of chemically competent BL21 (mscl-) E. coli. Prepare minimal medium and thin imaging coverslips. Transfect cells with pGFP and image/photograph the fluorescence (can we see them moving? Do we need a video camera?) Organize plasmids and cloning fragments. Determine schedules and communication strategy for cloning. Belinda and Juilia prepared minimal medium solutions. Aaron and Brock ran a gel on the digest for MSCL but there was no DNA seen. The Bio bricks are finally in!!!! Ashwin and Adam did midiprep of pGFP and got a nice pellet. Also did 2 small-scale seedings of MscL, one from Stable3 cells and the other from DH5a.

May 30: We will have an ORGANISATION MEETING at 12pm in the B floor conference room. Coffee, tea and cookies will be served. PLEASE be there, because we need everyone to help in keeping things organised. If you REALLY can't make it, I will send you minutes of the meeting, but it is critical to have everyone start out on the same wavelength.
EcoMscL seedings were good. Carried out 2 minipreps: seeding from DH5a (Ashwin, Adam, Alex) and Stable3 (Aaron, Jieun, Brock). Digested the Eco-MscL minipreps with XmnI and ran a gel. The bands are faint but at the right position (3888 & 1420). NB: for screening use 2-5uL of miniprep and a total of 15uL solution (we used 50uL => faint bands). Stable3 bands looked better than DH5a.

May 31: Julia and Adam met in the morning to discuss Team 1's project w/ Jay. Decided to focus on Fos and Jun plasmids. First experiment will be to try to transform those two plasmids together into the same bacterium. If Alex gets this message, he can go ahead and start this experiment. Alex, ask Jay for the two plasmids and then all it is just a transformation. Teams 2 and 3 did research on their individual projects. Designated a binder for each team's plasmids, papers, etc. Had a presentation on iGEM from Andrew and talked about some good project ideas and fundraising ideas.

May 31: (TEAM 3) Today Ashwini, Ashwin and Jieun did background research on luciferase and luciferin in the morning. We found out that luciferase (firefly luciferase, the plasmid vector available in the lab, and this is a monomeric protein unlike the bacterial luciferase that is a heterodimer) is too big for MscL channel. (The dimensions were approx 119.53 x 119.53 x 94.68 Angstrom while the maxium diameter of MscL is only 40 Angstrom.) Making luciferin getting into the cell through MscL was considered, yet this would be very inefficient since it is applied in the opposite direction of the mechanism of openig MscL channel. Instead, we decided to take on from the following idea: while luciferase is being expressed in the cell, we can change the environment (ie.pH change causes different colour emissions of luciferase) or we can allow small molecules to go through the cell membrane and work as a switch, per se. We were also interested in Elvis' suggestion from yesterday's meeting when he talked about controlling bacterial growth to formulate a "picture." (By combining the changing of colours and controlled shape of bacterial lawn, we could create a domino effect where the cells would light up/change colour due to the adjacent cell activities.) We also found out that the presence of luciferin is crucial for luciferase activity since it provides ATP catalysis and O2. As of now, we are still at finding related articles and information. - Jieun (12:14am June 01/06)

June 1:

• Team 3: Today we found an article on how osmotic shock affected one particular bacteria. If we are still continuing with the idea of osmotic shock, I think it would be a good reference article. Here's the link: http://www.blackwell-synergy.com/links/doi/10.1111/j.1432-1033.1997.00572.x/pdf

Some interesting websites to revisit http://www.jbc.org/cgi/content/abstract/275/9/6047 We were thinking of expressing luciferase and MscL in a Stable 3 cell, and then during the osmotic shock, as the water flows into the cell, there is a chance that luciferin will be able to enter the cell according to Elvis. So hopefully, the luciferin will then react with luciferase reslting in the emission of light. We think that this idea is worth a try. We found an article pertaining to the influx of solutes "Release of Thioredoxin via the Mechanosensitive Channel MscL during Osmotic Downshock of Escherichia coli Cells" by Bassam Ajouz, Catherine Berrier, Alexia Garrigues, Madeleine BesnardDagger , and Alexandre Ghazi§

• Team 3 (Cont'd)- We digested the pGFP vector with EcoR1 and Hind 111. The digestion was then run on a gel, the proper bands were obtained, and the vector was physically extracted.

• Elvis-Horia: Chemically Competent MJF367 & MJF465 with pUC19 cell plates were very good (bacteria lawn); seeded each strain. Transformed MJF367-pGFP & MJF465-pGFP and plated.

• Team 1: Today both the person responsible for half GFP's and the person responsible for sticky cells were e-mailed. No response fro Sticky cells and the GFP person was looking but did not find the plasmids. We were mostly researching today regarding a new Idea bout making a flagella fusion protein so it can attach to the membrane of another cell and forma a chain. We think that the only protein we can make a fusion of is the FliD. We were not successfull in asking the Microbiology departemtn about those fusion proteins, nor were we able to find this on the Internet as of current Date

• Team 2: Dissolved the BBa_I5610 in 10 uL of dH2O, and then transformed dh5α with the plasmid. Jamie, Adam, and Aaron prepared more LB, and Aaron plated the the dh5α onto a plate using 2 uL on one half and 20 uL on the other half.

June 2:

• 1. Elvis has done the seeding of the GFP bacteria from the plates with MJF367-pGFP & MJF465-pGFP and placed it in the incubator. After ~6h30 incubating, Horia added 1uL IPTG and placed in incubator for another hour. Update: The cells showed minimal Green Fluorescence under the UV wand. 2.The control of the MJF-pUC19 seeding was cloudy; the seeding were thrown away.

• Team 1: Because the person with the half GFP coluld not find the plasmids. We needed to get the mammalian plasmids from upstais to make a recombination to bacterial expression plasmids.

• Team 3: We extracted the GFP vector from the gel in the tube labeled "GFP by HIND 111 EcoR1 June 1,2006". The tube containing the purified DNA GFP vector from the gel is labeled "Gel extraction of GFP vector 6/2" A gel was then run to screen the GFP vector as well as the lucferase genes. Well #1 had the DNA ladder, Well #3 is the DNA from the "Gel Extraction of GFP vector 6/2", Well #4 has the "luciferase gene eluate May 10, 2006", Well #5 contains DNA from "lucif gene 18/06".

• The gel was analyzed and the GFP vector did not show up on the gel. However, both luciferase bands appeared at the appropriate site. The file was saved as "jun 2- GFP,luc gene" in the iGEM folder.

NB: Two dH2O bottles smelled like turkish toilets, so I threw them out and placed the remaining bottles under the pcr enclosure.

June 4 evening:

• Elvis seeded MJF465-pGFP cells in 1mL LB with AMP

June 5:

• Jieun diluted the MJF465-pGFP seeding into 5mL LB. After 4 hours, Horia verified O.D. with 1mL of culture to obtain a value of 0.25 and replaced them in the shaker-incubator. One hour later, 4uL of IPTG was added and left in S-I for Jamie to pick up later (~7:30pm).

• Team 1: We prepared competent cells Top 10F'. We have also ordered primers for the Jun-YFP and Fos-YFP.
• Team 3: We re-digested pGFP (from May 29th's midiprep) with EcoRI and HindIII. The gel was slightly smeared and the band for the GFP insert was very dim. But there was a visible band of pGFP vector (with no insert), which was cut out. The Gel Extraction procedure was then carried out, and the tube was labeled "Purified pGFP 06/05", and stored in the freezer in spot 1A of Cloning Fragments Box #1.