Part:BBa_K802003
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 low copy number plasmid in B. subtilis and a high copy number plasmid in E. coli.This plasmid is a derivative of pHT304 [2] in which the polylinker has been reconstructed to meet the IGEM standard. It has a polylinker containing all 4 of the iGEM restriction sites.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 6506
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6506
Illegal NheI site found at 3377
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 6512 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6506
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 6506
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 6506
Illegal XbaI site found at 6521
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3203
Illegal BsaI.rc site found at 5190
Characterization
Transformation yield
In E.coli NM522 strain, the average yield was 3.7 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 71 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_K802004, 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 BBa_K802003 part (the saturated culture and the 2 diluted solutions) was laid 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 bacterial cells.
BK37 stands for the NM522 strain transformed with BBa_K802003 (left side of each plate) and BK38 stands for the NM522 strain transformed with BBa_K802004 (right side of each plate)
As it can be seen in 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 are inoculated with bacteria containing the shuttle vectors BBa_K802003 and BBa_K802004. 3 samples per Ampicillin concentration were tested 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 :
BK37 stands for the NM522 strain transformed with BBa_K802003 (pBKL35) and BK38 stands for the NM522 strain transformed with BBa_K802004 (pBKH36)
Conclusion
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 the BBa_K802003 part or with the BBa_K802004 part was spotted on the plate.
35 stands for B. subtilis 168 strain transformed with BBa_K802003 (left side of each plate) and 36 stands for the B. subtilis 168 strain transformed with BBa_K802004 (right side of each 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 :
BK49 stands for the B. subtilis 168 strain transformed with BBa_K802003 (pBKL35) and BK50 stands for the B. subtilis 168 strain transformed with BBa_K802004 (pBKH36)
Between 0 and 1 mg/mL there is no significant difference between the two strains. However, starting from 1.1 mg/mL, there is a significant bacterial growth for BK50 strain as opposed to BK49 strain. As a result, the shuttle vector BBa_K802004 gives a higher resistance to Erythromycin than BBa_K802003.
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 pHT304. Each primer was designed 100 bp upstream (and downstream) the modified sequences. The sequencing confirmed the expected sequence for the iGEM polylinker and the deletion of the SpeI site from the Bacillus coding sequence.
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 containing 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 up to 0.9 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.
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