Team:BYU Provo/Team Thermosensor/Week19

From 2011.igem.org

Team BYU Provo

Home
Team
Project
Results
Protocols
Parts Submitted
Modeling
Safety
Attributions
Notebooks:
- Team OxyR:
  - The Beginning
  - Week 1
  - Week 2
  - Week 3
  - Week 4
  - Week 5
  - Week 6
  - Week 7
  - Week 8
  - Week 9
  - Week 10
  - Week 11
  - Week 12
  - Week 13
  - Week 14
  - Week 15
  - Week 16
  - Week 17
  - Week 18
  - Week 19
  - Week 20
  - Week 21
  - Week 22
  - Week 23
  - Week 24
  - Week 25
  - Week 26
  - Week 27
  - Week 28
- Team Thermosensor:
  - The Beginning
  - Week 1
  - Week 2
  - Week 3
  - Week 4
  - Week 5
  - Week 6
  - Week 7
  - Week 8
  - Week 9
  - Week 10
  - Week 11
  - Week 12
  - Week 13
  - Week 14
  - Week 15
  - Week 16
  - Week 17
  - Week 18
  - Week 19
  - Week 20
  - Week 21
  - Week 22
  - Week 23
  - Week 24
Indiana Trip
Outreach

Week 19 (Aug. 21 – 27)

Aug. 22

Plates left over weekend...not sure if TS is working properly colony 1 from griff med looks great, but no guarantee on the others.

With so few colonies 2nd transformation set up including a new one for griffshort

Griffshort annealing 53-5

10 uL IG59 griffshort for
10 uL IG64 griff short rev
ran on boil program
let stand for > 10 min.

Standard Ligation rxn 53-6

6.5 uL ddH2O
1.5 uL 10X ligase buffer
1 uL T4 DNA ligase
3 uL vector (pIG 15 gel)
3 uL inser (53-5 griffshort)

Set up Transformations for 51-6 (mutation 11), 51-7 (griff med) , 53-6 (griff short) and the purified plasmids TSA, I, and J.

Aug. 23

Mutagenic PCR – In order to get a wide range of different thermosensors we will be using a low fidelity PCR technique. Our template DNA will be from the purified plasmid TSA which contains a thermosensor designed to be similar a thermosensor found in listeria monocytogenes. As the PCR is carried out random nucleotides will be placed causing many different mutations of the TSA thermosensor. We will be using protocol similar to that found in the paper “Generation of synthetic RNA-based thermosensors” (T. Waldminghaus et. al.)

First created a CT/GA stock

10mM CT (20 uL) --> same for GA
2uL C 100mM
2uL T 100mM
16uL ddH2O
20 uL total = 10mM C/T

2mM CT --> same for GA
1uL 10mM C/T
4 uL ddH2O
5 uL C/T 2mM stock

Dilute MnCl2 10mM --> 5mM

40uL total

20uL MnCl2
20uL ddH2O

55-1 Master mix

108uL ddH2O
20uL pol. buffer
15uL C/T (10mM)
5uL G/A (2mM)
15uL primer IG54
15uL primer IG47
2uL Taq polymerase
20uL 5mM MnCl2
40uL of master mix in tubes 1-4

Added 1.5uL purified plasmid TSA to each PCR tube

55-2 Master Mix

108uL ddH2O
20uL pol. buffer
15uL GA 10mM stock
5uL CT 2mM stock
15uL primer IG54
15uL primer IG47
2uL Taq polymerase
20uL 5mM MnCl2
40uL of master mix in tubes 5-8

Added 1.5 uL purified plasmid TSA to each PCR tube

Ran PCR according to GB1 mutagenesis protocol

24 August 2011

Plated to plates from plates TSA, I and J to test for thermosensor activity. One plate incubated at 30’C overnight and the other at 37’C.

Results: All 3 have more LacZ expression at 37o . TSI has very minimal LacZ expression at 30o and J and A are mostly white.

Ran gel on mutagenic PCR 55-1 and 55-2.