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

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Cholera Detection March - April Notebook: April 1 - April 14 Daily Log




Overview
March-April
May-June
July-August
September-October

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.


3/21/13

- Checked up on results for 3.20 Phage Viability Test


3/22/13

- Discussed results from tittering experiment (preliminary experiment 1)

Contamination mostly likely resulted from the step involving suspending phage in LB – obvious contamination in the LB bottle
One of the stock phage solution had contamination as well

- Discussed step of attack with Dr. Grose

Decided to go with T7, if necessary Qbeta
Need to learn to make top agar at various concentrations
Need to do more background research and decide whether to assemble phage capsid in vitro (plasmid + E coli) or do direct mutagenesis of phage genome
Need to correspond with the isolation team

- Sequencing will be for individual genes to cut down cost

Need to design primers and get to know the genome of the phage

- Learnt about Mega5 to compare genome and protein sequence


3/25/13

- Reported on past week and plans for this week

From last week: titering experiment
This week
Learn to make top agar at various concentrations
Background research to determine in vitro assembly vs altering genome – look into specific techniques
Comparing genome of phage and decide on possible site-directed mutagenesis options

- Start working on designing our site directed mutagenesis

Qbeta vs MS2
Look for places where sequences are significantly different
Might be worthwhile to look at capsid structure to identify the regions where interactions between subunits take place
Qbeta vs T7 major
No real consensus – more worthwhile to compare capsid protein sequence of T7 with those that have similar size to it
T7 major vs minor
Minor is longer, but not necessary – the tail overhang is due to ribosome moving two codons downstream instead of three
Suggest we can direct mutation to the poly-U site and prevent ribosome slippage
Qbeta major vs minor
Just continue transcribing after it reaches the stop codon. What does the stop codon code for?

- Research into in-vitro assembly vs direct mutation of phage genome

It seems that we’ll need to clone the genome of the phage into a plasmid and let it assemble in an E coli
Using chemicals we can induce random mutations in phage – might be worthwhile if selection in agar is not working as well.


3/27/13

- More research on genome of enterobacteria phage

Generation of the major and minor capsid in Q beta
Capsid protein information research
Capsid protein sequence comparison

- Outlined protocol for producing stock top agar

4.3 Top Agar Stock Preparation


3/29/13

- Worked on our first team presentation.