Team:BYU Provo/Notebook/Cholera - Detection/Winterexp/Period3/Dailylog

4/1/13

-KK Our transformations worked this time and we had several beautiful lawns of E.Coli. The E. Coli did NOT fluoresce either red or green to the naked eye. We haven’t yet looked at them under UV light. Clarisse streaked several singles, and also set up two overnight colonies. Kelton and I practiced streaking to singles for the first time, using old plates. We placed our E.Coli singles in the 37 degree room to grow, along with the overnights on the shaker. Most of today we spent presenting our progress thus far. It appears that bacteriophage K139 will not be very useful to us, as we will need to use E.Coli bacteriophage lambda for our system. Dr. Grose has ordered Lambda and it is on it’s way. Our goal is to incorporate the Lambda genome as a lysogenic prophage into the genome of E.Coli, and have that prophage remain dormant until the presence of cholera’s autoreceptor triggers the phage to be excised from E.Coli’s genome and become lytic. The lysogeny/lysis balance is determined by the relative concentrations of two proteins, CRO and CI. The two proteins are mutually exclusive; if one is being expressed, then the other cannot be expressed, because they share a promoter region. Expression of CRO triggers the events that lead to replication and lysis. Expression of CI induces and maintains lysogeny. Our plan is clone the CI protein into E.Coli following the Qrr4 protein and the CRO protein following HapR.

- KP We started today with each group presenting their research and game plan for the coming weeks/months. It seems like we all know, or at least have a direction, to our next plan of action. Our E. coli turned out well and we were able to single out a colony in order to prepare overnight. After further research, it is clear that we will not be able to use T phages in our E. coli as a means to destroy cholera biofilm. We have all done more research on lambda, and it appears to be our best lysogenic phage choice. It is well studied and the mechanism for turning on the lytic cycle is very well understood.

4/3/13

- KK Not this Friday, but next Friday, we will have our Final Presentations. They will be done by sub-group; my sub-group is the cholera-phage approach. As part of the project, I need to attend a science seminar. Tomorrow I’ll attend the seminar by John Roth on something to do with molecular biology, then write a one-paragraph summary of HOW the presentation was done, the showsmanship of the presentation. On the day of our presentation we need to explain the background of what we have done, then go into our methods and results, then give our conclusions. Today we followed the mini-prep/Alkaline lysis procedure to purify plasmids from our overnight samples. We followed the step-by step protocol given in the kit we used. We chose to use 1 mL of our E.Coli overnight solution in our samples ... and at the end, our plasmid concentrations were low (24 ng/microL and 16ng/microL, respectively). We used the spectrophotometer in Dr. Grose’s lab to determine the concentrations of our plasmids. So, in the future, we ought to a) include Dr. Grose as we follow the protocol to ensure we are doing things correctly, and b) probably use more of the E.Coli sample to get more plasmids and hopefully a higher concentration.

- KP April Today we purified the plasmids and got them ready to be sequenced. We used Dr. Grose’s spectrophotometer. We learned how to analyze the graph in order to know if we just have DNA or if there is something else in our mixture after the purification process. The bump of our graph was right over 260, so we know that we successfully purified our plasmids! The only object of concern, is the fact that we had a very low concentration of plasmids. The instructions in the kit advised 1-3 mL of E. coli potion for high copy DNA. We used 1 mL, so next time we will try 2-3 mL to see if we can get a higher concentration. We are unsure if we did something wrong, if there is something wrong with the kit, or if we just need to use more of the E. coli overnight potion. I need to become more familiar with the sequence of our plasmid, and we need to find out why our E.coli doesn’t glow. When I understand the plasmid sequence, hopefully we can know what is missing or defective in our E. coli that keeps it from glowing.

4/5/13

- KP Today we made a plan of exactly what we need to do in the next week and coming months. Our biggest priority at this time is sequencing the plasmid from last years iGEM team. I was reading in the iGEM registry, and I saw that there is a pIG78 A and B. We tried A, but we haven’t tried B. On Monday we will try to transfer B into E. Coli and see if B works better than A. Next week our goal is to completely sequence the plasmid to see if something is missing or incorrect. I also need to do more research to understand better what last years team did and also the specifics of how we are going to set up our phage and how we are going to induce the lytic cycle in the presence of cholera.

- KK Today we set out some long-term priorities for what we should do from here on out. Our priorities: 1. Determine is the QS circuit working? We aren’t getting RFP right now … what’s wrong? a. We need to determine what primers were used to amplify the HapR and Qrr4 out of cholera b. What is the difference between pIG78 A and pIG78 B? There are two plasmids that are the same? We hope to KNOW the answers to these questions by Thursday 2. Clone CI behind Qrr4 promoter; determine where in plasmid and order primers order primers 3. Clone CRO behind HapR promoter DONE by Mid-May 4. Modify tails of phage (after C1 and CRO steps complete) DONE by summer 5. If possible, use selectable marker on phage to purify many E. Coli that have prophage incorporated into the genome. So these are the steps we set out, we need to discuss with Dr. Grose where we should be the C1 and CRO genes, because the plasmid is already very full. Personally, I need to understand the plasmid we’re using, the Lambda repression circuit, and the Lambda tail proteins better.

4/8/13

- KK The other cholera-subgroup is successfully growing biofilm. They have found that cholera biofilm grows best in a salty solution (their best imitation of seawater) at 37 C. Today we set up a quick experiment to see how our pIG78d+ E.Coli would respond to the presence of cholera. Using a cholera sample that was NOT growing much biofilm (the sample had been grown in normal LB at 30 C), we set up 5 test tubes. 1. 4 mL plain LB 2. 4 mL plain LB + 500 microL V.Cholerae solution 3. 4 mL plain LB + pIG78D DH5alpha E.Coli 4. 4 mL plain LB + 100 microL V. Cholerae solution + pIG78D DH5alpha E.Coli 5. 4 mL plain LB + 500 microL V. Cholerae solution + pIG78D DH5alpha E.Coli We set the overnight tubes in 30 C room on the shaker. Also, we set up two E.Coli overnights for miniprep on Wednesday. As part of our presentation on Friday we plan to present the results of whether our plasmid is functioning to detect cholera or not and why. We are still waiting for the sequencing results to come back to us. We also will present the research we have done on Lambda.

- KP I worked with kendall to set up an overnight so that we can do a mini-prep tomorrow to continue our sequencing of our plasmid. After we completed our overnight prep, we set up an experiment to test to ability of our plasmid to give off a color response to cholera. We prepared the 5 tubes that Kendall explained above, we then put them in the 30 C room overnight and we will check on them tomorrow.

3/22/13

- Discussed results from tittering experiment (preliminary experiment 1)