IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/11

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Working on E.coli trpR mutant

I have to test the mutant strain CY15001 (trpR-) that Dr. Charles Yanofsky kindly provided us.

As the mutation consists of a frame shift, I´m going to make a PCR reaction and then sequence the amplified products.

The primers that I am using are:

Forward (5'->3'): CGC ACG TTT ATG ATA TGC TAT CG

Reverse:(5'->3'): AGG CCT ACA AAA TCA ATC G

These primers would amplify trpR coding region (326nt), 87nt upstream from the start codon and 12nt after the stop codon. Thus, obtaining a final PCR product of 426nt long.

Experimental Setup

PCR colony: Protocol

1. Take with a toothpick a sample of the colony.

2. Dissolve the sample in 200μL of Tris-ETA 10/1-NaCl 10mM solution.

3. Heat the sample during 10 min at 95°.

4. Centrifugate at 14 000 rpm during 2 min.

5. Take 10μL as DNA template for PCR reaction.

  • PCR Mixture:
Reactive Quantity
MgCl2 2.5μL
Buffer 10X Taq 5μL
dNTP's 2.5μL (0.4mM)
Primer Forward 2.5μL (5pmol/μL)
Primer Reverse 2.5μL (5pmol/μL)
DNA template 10μL
Taq polymerase 1μL
HPLC Up to a final volume of 50 μL of the mixture

  • 35 PCR programmed cycles:

The reaction starts at 95°C during 5 min.

Each cycle is programmed as follows:

Temperature Time
94°C 45s
55°C 45s
72°C 45s

After the cycle 35, the temperature changes to 72°C during 5 min and ends at 4°C.

  • Gel preparation:

1. Use 0.8% agarose mixture.

2. Melt the agarose for 4 min at power of 30, inside the microwave.

3. Wait to withstand the agarose temperature by yourself.

4. Put the mixture on the electrophoresis camera device. And wait for 5-10 minutes to get solidified agarose.

  • Samples load:
Reactive Quantity
Dye 1 μL
Ladder 1μL
PCR sample 10% from the total volumen

  • Electrophoresis parameters:
Voltage Running Time
80 50min

  • Gel stain:

1. First wash: Ethidium bromide + water under shaking during 10 minutes.

2. Second wash: Water under shaking during 10 minutes

Reactive Quantity
Ethidium bromide 100 μL

Results:E.coli trpR mutant

Samples:The trpR mutant colonies that were selected to make the colony PCR were: 1,5,8.

Colony control: E.coli wt

I failed doing all colony PCRs. I didn´t get any amplified products, the electrophoresis gel was empty in each respective PCR colony lane. I will repeat the experiment.

Working on LovTAP promoters:Transformation

In order to test the new LovTAP, designed considering Dr. Devin advices and Lausanne team modeling results. I had to choose the proper promoters under which LovTAP expression will be regulated.

After looked up the promoter in the registry of biological parts. I decided to use the following members of the family J23:

Strength Entry Plate Well
Strong J23102 2010_1 18G
Medium J23110 2009_1 20C
Low J23117 2010_1 20O

All these promoters are contained inside J61002 plasmid, and control the expression of RFP protein. Besides, the plasmid harbors an ampicillin resistance.

Experimental Setup

1.Get the plasmids harboring each promoter, from the corresponding plates.

2.Transform cells.

3.Grow up the transformed cells in LB medium with ampicillin antibiotic. This is because the plasmids harboring each promoter have a resistance against ampicillin.

Reactive Quantity
Ampicillin 1μL for each LB medium mL

Results:LovTAP promoters

  • Promoter J23102 was successfully transformed. Six colonies produce RFP protein. I will extract the plasmids from three of them.
  • Promoter J23110 was not transformed. I will repeat the experiment.
  • Promoter J23117 seems to be transformed. From the three selected colonies (4,5 and 6) none produce RFP protein; maybe the production is very low. The colonies will continue growing at room temperature. I selected 6 new colonies (7,8,9,10,11 and 12) to see if they produce RFP protein, they were incubated at 37º C overnight and then were exposed at room temperature to continue growing.

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