IGEM:IMPERIAL/2008/New/Major Results/Transformation

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Transformation of B.subtilis

In order to expand the use of B.subtilis as a potential chassis we investigated the different protocols used for the transformation of B.subtilis. B.subtilis is used as the model for gram positive bacterium and have been extensively studied. A variety of protocols have been developed for the transformation of B.subtilis. We wished to investigate two different protocols, one relying upon natural competence of stressed B.subtilis and the using electroporation.


Transformation Protocol 1

Click this link for full Transformation protocol 1

Brief Description of Protocol

Prepearation of Competent cells:

  • Start an overnight culture,
  • Dilute overnight culture to an O.D.600 of 0.5,
  • Grow to an O.D.600 of 2.0-2.5,
  • Dilute cells and grow further in fresh media,
  • Freeze these cells,
  • Defrost cells and incubate cells with DNA,
  • Plate the cells onto a LB agar plate with suitable antibiotics.

Results

  • We performed a transformation using the integration plasmid pDR110. The competent cells were grown to an O.D.600 of 2.0 and transformed with 0, 40ng and 400ng of DNA. Transformants were selected with the antibiotic streptinomycin, the resistance of which is encoded by the pDR110.
  • The results showed that the B.subtilis transformed with no DNA yielded no transformants and B.subtilis transformed with 40ng and 400ng yielded about 10 transformants.
  • The protocol was carried out with two repeats and it was noticed that similar numbers of transformants were yielded for both repeats.

Transformation Protocol 2

Click this link for Transformation protocol 2

Brief Descrption of protocol

Preparation of Competent Cells:

  • Start an overnight culture,
  • Dilute overnight culture,
  • Grow to an O.D.600 of 1.5-2.0,
  • Freeze these cells,

Transformation:

  • Defrost cells and electroporate with DNA,
  • Plate the cells onto a LB agar plate with suitable antibiotics.

Results

  • We performed a transformation using the integration plasmid pDR110. Two repeats were carried out using this protocol. For the two repeats the competent cells were grown to an O.D.600 of 1.5 and 1.8 and transformed with a range of DNA concentrations from 0 to 400ng of DNA. Transformants were selected with the antibiotic streptinomycin, the resistance of which is encoded by the pDR110.
  • The results from this protocol were mixed, for the first repeat there were transformed colonies on the plate of B.subtilis transformed with no DNA. The most likely cause for this is that there was a contamination in preparation of these samples. For the second repeat no transformants was seen for the B.subtilis transformed with no DNA, showing that there was no contamination.
  • Comparison of the different efficiencies of transformation between experiments showed that the number of transformants was highly variable. The cause for this is thought to be the settings for electroporation equipment. We able to control the voltage and the resistance but not the pulse length. Previous studies have shown that a pulse length below 5 msec gives low efficiency of transformation. A maximum efficiency is found for a pulse legnth of 10 msec. Between our two repeats we found a range of pulse lengths between 3-11 msec and as a result variability in the number of transformed B.subtilis.


Verifying Integration

To test whether integration was succesful, single colony PCR was used with various primers specific to sequences within the B.subtilis genome and the pDR111 vector.

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