Team:BYU Provo/Team Thermosensor/Week19

From 2011.igem.org

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==Week 19 (Aug. 21 – 27)==
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===Aug. 22===
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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.
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===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.)
 +
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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===
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 +
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.
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Latest revision as of 08:57, 24 September 2011

Team BYU Provo

BYU Provo
 

Contents

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.

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