Team:VIT Vellore/Results

Team VIT Vellore/Description

The results of the experiments performed

Introduction

Our final assembled construct contains resistance against Chloramphenicol (pSB1C3, backbone). The inserted fragment contains antisense sequence against Ampicillin resistance gene present in pBR322. pBR322 contains resistance against Ampicillin and Tetracycline.


Bacteria are transformed with:

  1. pBR322, conferring resistance to Ampicillin
  2. Assembled construct conferring resistance to Chloramphenicol
  3. Double transformants containing both pBR322 and our assembled construct. The bacterial colonies can survive Tetracycline and Chloramphenicol antibiotics but are sensitive to Ampicillin (due to the antisense sequence against Ampicillin resistance present in our construct).

Assembly of the required construct

The linearized backbone, pSB1C3 2070 bp, is ligated to our insert (designed composite fragment) (483 bp) following Restriction Digestion (using XbaI and SpeI). The insert was initially amplified using PCR. Agarose Gel Electrophoresis was performed to confirm the assembly of the desired plasmid.

PCR Amplification of part

Confirmation of successful cloning


Transformation Results

Transformation of Ampicillin resistant pBR322 into E. coli cells


E. coli cells were transformed with pBR322 plasmid containing Ampicillin and Tetracycline resistance and plated on LB plates containing Ampicillin. Control plate had non-transformed E. coli competent cells on Ampicillin-containing plates.

Results:

  1. Test plates showed bacterial growth.
  2. No growth observed on control plate.

Inference:

  1. E. coli cells do not have Ampicillin resistance natively, therefore control shows no growth.
  2. Incorporation of pBR322 plasmid into E. coli cells confers them with Ampicillin resistance. Therefore, cells transformed with pBR322 survive Ampicillin.
  3. Conferring Ampicillin resistance to E. coli enables us to further validate our construct having antisense against the Ampicillin resistance gene.

Transformation of our construct (containing Chloramphenicol resistance and antisense against Ampicillin) into E. coli cells


E. coli cells were transformed with our construct containing Chloramphenicol resistance and plated on LB plates containing Chloramphenicol. Control plate had non-transformed E. coli competent cells on Chloramphenicol-containing plates.

Results:

  1. Test plates showed bacterial growth.
  2. No growth observed on control plate.

Inference:

  1. E. coli cells do not have Chloramphenicol resistance natively, therefore control shows no growth.
  2. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance. Therefore, cells transformed with our construct survive Chloramphenicol.
  3. Conferring Chloramphenicol resistance to E. coli enables us to confirm that our construct (containing antisense to Ampicillin resistance gene) has been successfully incorporated into the E. coli cells.

Transformation of E. coli cells with our construct (containing Chloramphenicol resistance and antisense against Ampicillin) + pBR322 (containing Ampicillin and Tetracycline resistance)


E. coli cells were transformed with our construct (containing Chloramphenicol resistance and antisense against Ampicillin resistance gene) along with pBR322 (containing Ampicillin and Tetracycline resistance) and plates on LB plates containing Chloramphenicol and Tetracycline. Control plate had non-transformed E. coli competent cells on Chloramphenicol and Tetracycline containing plates.

We refer these cells as ‘double transformants’ in further experiments.

Results:

  1. Test plates showed bacterial growth.
  2. No growth observed on control plate.

Inference:

  1. E. coli cells do not have Chloramphenicol and Tetracycline resistance natively, therefore control shows no growth.
  2. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance and incorporation of pBR322 into the E. coli confers them with Tetracycline and Ampicillin resistance. Therefore, cells transformed with our construct along with pBR322 survive Chloramphenicol as well as Tetracycline.
  3. Cells surviving on these plates have our construct having the antisense to the beta-lactamase (Ampicillin resistance) gene present in pBR322.

Validation of our Construct

Plating double transformants on (Amp + Tet + Chl) plates


Plating double transformants on (Amp + Tet + Chl) plates:

The double transformants containing pBR322 (containing Ampicillin and Tetracycline resistance) along with our construct (containing Chloramphenicol resistance and antisense against Ampicillin resistance gene) were picked up from the previous Chl+Tet plate and were plated on plates containing Ampicillin, Tetracycline and Chloramphenicol.

Control plate had non-transformed E. coli competent cells on Ampicillin, Tetracycline and Chloramphenicol containing plates.

Results:

  1. Low growth observed on test plate.
  2. No growth observed on control plate.

Inference:

  1. E. coli cells do not have Chloramphenicol, Tetracycline and Ampicillin resistance natively, therefore control shows no growth.
  2. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance and incorporation of pBR322 into the E. coli confers them with Tetracycline and Ampicillin resistance. Therefore, cells transformed with our construct along with pBR322 should survive Chloramphenicol as well as Tetracycline. But, since antisense is also incorporated as a part of our construct which inhibits the Ampicillin resistance gene present in pBR322, thereby resensitizing to the bacteria to amplicillin. Therefore, there is very low growth due to Ampicillin present in the plates and the inhibited Ampicillin resistance gene.

Growing double transformants in LB broth under selective pressure and plating them on (Chl + Tet) and (Amp + Chl + Tet) plates


The double transformants containing pBR322 (containing Ampicillin and Tetracycline resistance) along with our construct (containing Chloramphenicol resistance and antisense against Ampicillin resistance gene) were inoculated in LB broth containing Chloramphenicol and Tetracycline (selective pressure).

The cells from the broth were then plated on:

  1. Chloramphenicol + Tetracycline (Chl + Tet) containing plates
  2. Chloramphenicol + Tetracycline + Ampicillin (Chl + Tet + Amp) containing plates

Results:

  1. Growth observed on (Chl + Tet) plate was higher than the initial growth observed after transformation on (Chl + Tet) plates.
  2. Low growth was observed on the (Chl + Tet + Amp) plate.

Inference:

  1. LB broth gives selective pressure for the double transformants (containing our construct + pBR322) to retain both the plasmids.
  2. Sub-cultured bacteria containing our construct along with pBR322 are more pure when replated on (Chl + Tet) plates. Therefore, the growth observed is higher on cells plated after sub-culturing in the (Chl + Tet) plate when compared to the growth observed on the initial (Chl + Tet) plate.
  3. Low growth is observed on (Chl + Tet + Amp) plate indicates successful integration of our construct containing antisense to Ampicillin resistance which inhibits the Ampicillin resistance gene, thereby, preventing growth in presence of Ampicillin.


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