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Team:BYU Provo/Notebook/Cholera - Detection/Winterexp/Period3/Dailylog
From 2013.igem.org
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<font size="4"> '''4/1/13''' </font> | <font size="4"> '''4/1/13''' </font> | ||
| - | + | 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. | |
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| + | 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. | ||
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Revision as of 21:49, 5 June 2013
| Cholera Detection March - April Notebook: April 1 - April 14 Daily Log
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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 - Learned about how to start an overnight liquid culture for host bacteria - Learned about how to create bacterial glycerol stock - Made fresh LB and concentrated top agar stock
4/4/13 - T7 arrived - Overnight liquid culture for bacterial host started by Dr. Grose
4/5/13 - Background research on phiX174: the only phage we have in stock - Performed 3.20 Phage Viability Test
3/21/13 - Checked up on results for 3.20 Phage Viability Test
3/22/13 - Discussed results from tittering experiment (preliminary experiment 1)
- Discussed step of attack with Dr. Grose
- Sequencing will be for individual genes to cut down cost
- Learnt about Mega5 to compare genome and protein sequence
3/25/13 - Reported on past week and plans for this week
- Start working on designing our site directed mutagenesis
- Research into in-vitro assembly vs direct mutation of phage genome
3/27/13 - More research on genome of enterobacteria phage
- Outlined protocol for producing stock top agar
3/29/13 - Worked on our first team presentation.
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