NB: BB12mut is the ligation product of BB1 + P2, and BB123mut is the ligation product of BB12 + P3, therefore it is the complete biosensor.

Objectives

The overall purpose is to check if the bacteria obtain from the transformation with BB12mut and BB123mut contain the good genetic constructions.

Materials

Bacteria tansformed with BB12mut and BB123mut (made on 05/09/16)
Primers: A12 (forward) and A13 (reverse)

Protocol

PCR

1. 2 Mix for 17 samples (Total volume of each Mix : 850µL), in an Eppendorf tube :

705.5 µL H2O

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

17 µl Primer A12 (1 µM final)

17 µL Primer A13 (1 µM final)

17 µL dNTP (200 µM final, NEB #N0447S)

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

Add in 32 PCR tubes, in the respected order:

50 µL Mix

One colony from the different petri dishes (pick one colony, put some on a new plate, divided into squares, and put the remaining bacteria into the PCR mix)

Gently mix the reaction

2. Short spin centrifugation

3 Set the following parameters for the PCR reaction :

P12 (2.195 kb)
Lid température 95°C
Initial denaturation : 95°C, 5 min
30 cycles of : 95°C, 30 s
58°C, 60 s
68°C, 2 min 12 s
Final extension : 68°C, 5 min
Hold : 4°C

P123 (3.4 kb)
Lid température 95°C
Initial denaturation : 95°C, 5 min
30 cycles of : 95°C, 30 s
58°C, 60 s
68°C, 3 min 24 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.