Objectives

The overall purpose is to create PA (Pr - RBS - XylR - Term) and PB (Pu - RBS - Gaussia - Term) from our biosensor.

Materials

DNA fragments : BB123mut-6 (from mini prep 07/09)
Primers: A12 (forward) and BBA-R (reverse) for PA, and BBB-F (forward) and A13 (reverse) for PB.

Protocol

PCR

1. Mix for 5 samples (Total volume of Mix: 230 µL), in an Eppendorf tube:

   • 198.75 µL H2O

   • 25 µL Buffer Taq (1 X final, NEB #B9014S)

   • 5 µL dNTP (200 µM final, NEB #N0447S)

   • 1.25 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)

2. Add in 4 PCR tubes, in the following order:

   • 46 µL Mix

   • 1 µL primer forward (A12 or BBB-F)

   • 1 µL primer reverse (BBA-R or A13)

   • 2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)

3. —> Gently mix the reaction and perform a short spin centrifugation

4. Set the following parameters for the PCR reaction :

   • PA (2285 bp)

   • Lid temperature: 95°C

   • Initial denaturation : 95°C, 30s

   • 30 cycles of :

     • 95°C, 30 s

     • 58°C, 60 s

     • 68°C, 2 min 17 s

   • Final extension : 68°C, 5 min

   • Hold : 4°C

   • PB (1037 bp)

   • Lid temperature: 95°C

   • Initial denaturation : 95°C, 30s

   • 30 cycles of :

     • 95°C, 30 s

     • 58°C, 60 s

     • 68°C, 1 min 02 s

   • Final extension : 68°C, 5 min

   • Hold : 4°C

Electrophoresis: for screening the PCR results

1% Agarose gel:

1. Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL

2. Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid.

3. Add 5 µL of Gel Red 10,000 X (0.5 X final)

4. Flow the gel and place the combs

5. Wait until it is solidified. Remove slowly the combs.

Drop-off:

1. Short Speed centrifugation of samples

2. Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample

3. Drop-off 10 µL of Purple ladder and 12 µL of each samples.



4. Run at 90 V.

Mini-culture: bacteria transformed with BB12mut and BB123mut

16 Mini-cultures of bacteria transformed with BB12mut (3, 4, 5, 7, 8, 9, 11, 12) and BB123mut (1, 2, 4, 5, 7, 10, 11, 13).
Put the colony with satisfying PCR results from the plates divided into squares to a 50mL Falcon tube containing 5mL LB+Cm.

Results

1st electrophoresis: Expected results / Obtained results



2nd electrophoresis: Expected results / Obtained results



Interpretation

We obtain the desired strip for BB12mut, for all the colonies (n°1 to 14). As shown on the gel above, the strips are closed to 2.2 kb, which is the size of P1+P2. A sequencing is necessary to be sure of the obtained biobrick.

We obtain the desired strip for BB123, for all the colonies except for n°7. As shown on the gel above, the strips are closed to 3.4 kb, which is the size of P1+P2+P3. A sequencing is necessary to be sure of the obtained biobrick.



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