Team:BYU Provo/Notebook/Cholera - Detection/Winterexp/Period2/Dailylog

Cholera Detection March - April Notebook: April 1 - April 15 Daily Log

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.

-CH Presented more on our project goals for breaking down biofilm with phage. We will have to use lamda phage for our project because it has a lysogenic phase.

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.

-CH Set up mini prep from the overnight culture. Followed the instructions found in the instruction manual for the kit. The two samples we did gave us concentrations of 24ug/ul and 16ug/ulw which was low. Next time we need to pellet more than 1ml of saturated culture for the initial step. Also we could have done the ethanol wash spin down for longer. Submitted 2 samples for sequencing using the more concentrated mini prep and primers IG11 and IG12.

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.

-CH Discussed project goals.

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.

-CH Set up transformations with pJG78a (inserts2,5,3,6,4,7,1), pJG79a (2,3,4,5,6), pJG80a (2,3,5,6), pJG81a (2,3,5), and a control (no plasmid added). METHOD: 1ul plasmid prep added to 25ul of purified plasmid on ice 5mins, heat shock 1 min, recover on ice 3min, add 1ml of LB then put at 37 for 30 mins, plate 50ul of each sample on LB-Amp, put at 37 overnight. RESULT (4/9/13): control plate was empty. Lots of colonies from the other plates. So many I could bearly find a single isolated colony. The transformation from pJG81a was a nice green. All of the other samples showed little or no green fluorescence. Will have to set up more transformations with all the other stepping stone plasmids to see if anything will have RFP expressed. I also mapped out how the completed construct should look on paper with all the inserts. I did this using the information on the QS primers document. If everything is cloned in using the exact primers and following the sequences provided then the GFP protein was cloned in in the reverse direction and HapR was cloned in without a promoter.

4/10/13

- KK Results from our overnight cholera preps: The first three tubes from the left are controls, one with pure LB, one with cholera + LB (which was grown at 30 degrees by the other subgroup), and one with E.Coli + LB, respectively. The fourth and fifth tubes have cholera, E.Coli, and LB. The fourth has a slightly greater number of cholera (500 microL of cholera-saturated broth) compared to the fifth (100 microL of cholera-saturated broth). These are the results after two days of shaker-incubation at 30 degrees C, when the cultures are viewed under UV light. We went over the quorum-sensing circuit with Dr. Grose. The Cqss membrane receptor phosphorylates Lux U, which cascades phosphorylation to LuxO. LuxO, when phosphorylated, binds to the Qrr4 promoter, activating transcription of Qrr4 mRNA. Qrr4 mRNA interferes with HapR, so there is no protein HapR. HapR is a repressor whose binding site is at the promoter for GFP. When Qrr4 eliminates HapR, there is no repression of the expression of GFP. Thus, GFP should be ON when Cholera is NOT present. The reverse is also true. In the presence of Cholera autoinducer, CQSS acts as a phophatase on LuxU, which does the same to LuxO, which does NOT activate Qrr4 mRNA which does NOT degrade HapR which represses transcription of GFP. Thus GFP should NOT glow in the presence of cholera. Our results in these test tubes aren’t exactly in agreement with what we want.

- KP Today we worked on our presentation for Friday and we also went over the quorum sensing with Dr. Grose.

-CH We worked on our final presentation for this semester. I am focusing on the quorum sensing system in Cholera and how we have modified it in E.coli. There is still some confusion on the HapR responsive promoter the other students added to the modified QS. Is it just the HapR promoter? Is it negatively or positively responsive to HapR? HapR represses its own promoter. (see Regulatory targets of quorum sensing in Vibrio cholerae: evidence for two distinct HapR-binding motifs Amy M. Tsou1, Tao Cai2, Zhi Liu1, Jun Zhu1,* and Rahul V. Kulkarni3)

4/12/13

- KP We had our presentation today. It was good practice and I learned some things to improve on in the future. There was some confusion on the quorum sensing, because we thought that HapR was a repressor and that it shouldn’t glow green in the presence of cholera. It turn out that it is the other way around and that it should glow green in the presence of cholera and red in the absence of cholera. That still doesnt explain why our tests are glowing green in the absence of cholera. We will have to work backwards and find out exactly what it is that we have. I feel that we have a clearer view of what we need to do. Also, I need to do more research other than wikipedia on lambda phage and its lytic and lysogenic cycles, because in our presentation, I said that that lambda enters the lytic cycle when it is in a cell that is healthy and lysogenic cycle when the cell is in trouble. That is what it says on wikipedia. Anyway, when I said that, I was told that I was wrong and that it is the other way around. I will look into that. We also hopefully got our quorum sensing system worked out now. All we need to do (once we figure out what we actually have in our plasmids) is add the cro transcription site after the GFP, and in the presence of cholera more cro will be produced and lambda will enter the lytic cycle!

- KK Today was our presentation in class. This is the link to our powerpoint presenation: https://docs.google.com/presentation/d/14mUxVrUdTvNXaPGifXZs_lpXeMrFd-4CxSZ9o1bPqYo/edit#slide=id.p Our presentation gave a background for our project - though we ought to have been clearer WHY we want to use phage - and also the work that we’ve accomplished thus far in our group. We went a long time over in our presentation; it took a long time to explain the quorum sensing circuit, Lambda, and other things. Dr Grose sent an email with several suggestions for presentations in the future. They include being more concise with our time management and better coordinating within our group so that we don’t end up repeating ourselves. In the future we will be expected to be more polished presenters.

-CH Presented on the Cholera quorum sensing system and our plans for our project (ie, E.coli, lambda phage etc).

4/15/13

- KP Today we started out by getting photographed. We also watched the phage group’s presentations. It seems as if they have a good start to their project. We didn’t have too much time to do our own experiments, but we did set up an overnight to do mini-prep tomorrow, and we streaked out our plates of lambda phage. - KK The three phage groups presented today. I realized how very quickly each of our groups has specialized to the point that I have to concentrate deeply to understand what seems to be second nature to members of the phage group. That’s good! It means each of our groups is moving along nicely. The objective of the phage project is to make a library of sizes - the biggest and smallest phage capsids possible using phages that have already been well characterized and approved for medicinal purposes. One group is working on selecting for the largest phage possible, one for the smallest, and another group is developing a method to purify both phages as they are made. Afterwards, we laid out singles of our three E.Coli strains with Lambda prophage incorporated into its genome. Tomorrow Kelton and I are going to go in and check on them after our Intro to Medicine class.

-CH Listened to presentations from the phage evolution groups. Seems to me they are all doing well and have good plans for their projects. We also as a group figured out what we are going to do inbetween semesters. Meet this Weds at 5pm to set up some more transformations and redo the mini prep. Meet on Friday at 2pm with Dr Grose for help with making primers.

CH 4/17/13 Set up transformations for pJG78b, pJG79b, pJG79c, pJG80b, pJG81b, pJG81c, pJG81d. Method: Added 1ul of mini prep of each plasmid to 25ul of dH5alpha. 5min on ice. 2 min heat shock at 42. 2 min recovery on ice. 30min recover w/ 1ml of LB at 37. Plate 50ul onto LB-Am. 37 overnight. Result: Everything transformed ok but nothing had any signs of RFP.

CH 4/19/2013 Met with Dr Grose to design more primers. See primer doc BI247-BI252. These are to help with Cro, hapA, and more sequencing.