Difference between revisions of "Part:BBa K802004"

Latest revision as of 20:51, 26 September 2012

Shuttle vector for E. coli and B. subtilis

This part is a shuttle vector of 6.5kb which is Ampicillin resistant in E. coli and Erythromycin resistant in B. subtilis. It is a high copy number plasmid in both B. subtilis and E. coli. This plasmid is a derivative of pHT315 ^[2] in which the polylinker has been reconstructed to meet the IGEM standard. The polylinker contains all 4 of the iGEM restriction sites.

Characterization

Transformation yield

In E. coli NM522 strain, the average yield was 3.3 x 10⁶ cells/µg.The transformed cells were selected on LB medium supplemented with Ampicillin at a concentration of 100µg/mL.

In B. subtilis 168 strain, the maximum yield was 80 cells/µg. The transformed cells were selected on LB medium supplemented with Erythromycin at a concentration of 15µg/mL.

Antibiotic resistance

Given the fact that this shuttle vector is very similar to BBa_K802003, the two of them were tested in parallel. Two experiments were made in order to characterize the antibiotic resistance of the shuttle vectors : the first one on Agar plates and the second one in liquid cultures. In both cases the medium was LB supplemented with Ampicillin or Erythromycin at different concentrations.

The shuttle vector was transformed into NM522 strain in order to be tested.

A) Agar plates
Saturated cultures containing the shuttle vectors were diluted 10, 100, 1,000 and 10,000 times. A volume of 5 µL of each solution containing the strain with the part BBa_K802003 (the saturated culture and the 4 diluted solutions) was spoted on the plate. The volume of the cultures containing the second shuttle vector (BBa_K802004) was adjusted regarding OD₆₀₀ in order to spot approximately the same number of bacteria.

As it can be seen on the picture, at 10 mg/mL (i.e. 100 times the usual selective concentration) there is bacterial growth, even for the culture diluted 10,000 times (the corresponding inoculum is indicated by the number 4 written on the plates on the picture)

B) Liquid cultures

Multiple tubes containing LB medium and Ampicillin at different concentrations ranging from 0 to 3 mg/mL were inoculated with bacteria transformed with the shuttle vectors BBa_K802003 or BBa_K802004. The cultures were run in triplicate per Ampicillin concentration for each vector. As a negative control an Ampicillin sensitive strain was used to inoculate a solution of LB medium at the usual selective concentration of 100 µg/mL. The results are shown below :

To sum up, there is no significant difference concerning the Ampicillin concentration tolerance between the two strains, therefore between the two shuttle vectors as far as E. coli strains are concerned. At 2.4 mg/mL there is no bacterial growth for either strain.

The shuttle vector was transformed into Bacillus subtilis 168 strain in order to be tested.

A) Agar plates
Saturated cultures containing the shuttle vectors were diluted 100 and 10,000 times. A volume of 5 µL of each solution containing the strain with BBa_K802003 part or with BBa_K802004 part was spotted on the plate.

As it can be seen on the picture, B. subtilis transformed with BBa_K802004 grew perfectly well even for the culture diluted 10,000 times(the corresponding inoculum is indicated by the number 4 written on the plates on the picture). In contrast, B. subtilis transformed with BBa_K802003 shows antibiotic sensitivty for the most diluted cultures (10,000 x) at the four tested Erythromycin concentrations.

B) Liquid cultures

Multiple tubes containing LB medium and Erythromycin at various concentrations ranging from 0 to 1.5 mg/mL were inoculated with bacteria containing the shuttle vectors BBa_K802003 or BBa_K802004 and incubated 20 hours at 37°C. OD₆₀₀ of the cultures was measured and the value was converted in cfu/mL^[1]. The results are shown below :

Sequencing of the modified regions

The two modified regions of the shuttle vector were verified by sequencing. The primers were designed using the sequences from the original plasmids that were used to construct the shuttle vector pHT315. Each primer was designed 100 bp upstream (and downstream) the modified sequences. The sequencing confirmed that the site SpeI was eliminated. However, there is still yet to confirm the iGEM polylinker, even though the gel electrophoresis confirmed the existence of all 4 of the iGEM sites.

Usage and Biology

It can be used to transform E. coli and B. subtilis strains. However, it is highly recommended to amplify the plasmid before transforming a B. subtilis strain because the transformation yield is low compared to E. coli.

Functional Parameters

The tests showed that in E. coli NM522 strain, there is bacterial growth in LB medium having an Ampicillin concentration up to 1.2 mg/mL. Concerning B. subtilis 168 strain, the bacteria transformed with this plasmid could be selected with Erythromycin concentrations as high as 1.2 mg/mL.

References

^[1] http://bionumbers.hms.harvard.edu//bionumber.aspx?id=105453&ver=1
^[2] Construction of cloning vectors for Bacillus thuringiensis. Arantes O. and Lereclus D. (1991) Gene 108:115-119.