Team:Toulouse France/Description

iGEM Toulouse 2016

Backbones description

Backbone

The use of replicative plasmid in Bacillus subtilis is not as usual as in E. coli. Only one of such plasmid is described in the iGEM registry (pSB_BS0K-P from iGEM Munich 2012; BBa_K823026 and its slightly modified form BBa_K1351040). Since none of them were available from the registry, we eventually got BBa_K1351040 from ex-Munich iGEMers (thanks guys!). However, this plasmid is already huge (9358 bp), and we needed to insert great fragments inside. Our first attempts to clone in BBa_K1351040 failed. We therefore decided to operate a reduction of the plasmid size.

pSB_BS0K-Mini: new part BBa_K1937001

We reasoned that most of the pSB_BS0K-P plasmid content were of no need to our project (lacY, rfp, lacI…). Since supressing these elements also suppressed the suffix and prefix, primers with new suffix and prefix were designed to amplify the needed regions (selection markers and replication origins; figure 1). Nhe1 restriction site was also added at the 5’ end of each primers to circularize the PCR product.

Figure 1: reduction of the pSB_BS0K-P plasmid. Position of the primers are indicated by the blue arrow on pSB_BS0K-P (left part of the figure). The resulting PCR fragment is the blue pointed line. After digestion by NheI and self-ligation, the resulting pSB_BS0K-Mini plasmid was obtained (right part of the figure).

The new plasmid size is smaller by 3604 bp. It was purified from E. coli and used to successfully transform B. subtilis. Integrity of the whole sequence was assessed by sequencing using 10 primers distributed all along the sequence. This new plasmid was then used to create most of the Bacillus subtilis parts of our project, demonstrating its efficiency as a new backbone for Bacillus based projects.

OriKan (new part BBa_K1937002): and they all became Bacillus plasmids…

With the success of the pSB_BS0K-Mini, we decided that we should try to go even further by isolating what is the very essence of this replicative Bacillus plasmid: its Bacillus repU origin and its kanamycin resistance gene. We amplified the region containing these two elements with primers carrying the iGEM suffix and prefix (figure 2).

Figure 2: creation of the OriKan cassette. Position of the primers are indicated by the blue arrow on pSB_BS0K-P (left part of the figure). The resulting PCR fragment is the blue pointed line. After digestion by EcoRI and PstI and ligation in the pSB1C3 plasmid, the resulting pSB1C3-OriKan plasmid was obtained (right part of the figure).

The fragment was then sub-cloned in the pSB1C3 plasmid (between the EcoRI/PstI restriction sites). We checked the capacity of this new pSB1C3-Orikan plasmid to maintain in Bacillus subtilis and we were delighted to obtain clones. The actual presence of pSB1C3-Orikan in this colonies was assessed by PCR (figure 3). The sequence integrity of the OriKan cassette was also verified.

Figure 3: validation of the pSB1C3-Orikan presence in B. subtilis. PCR on colonies was performed using primers hybridizing in the kanamycine resistance gene and in the suffix. The colonies were issued from the transformation of B. subtilis by pSB1C3-Orikan (assays), by the pSB_BS0K-P plasmid (negative control), or from the transformation of E. coli by pSB1C3-Orikan (positive control).

These results demonstrate that the OriKan biobrick is sufficient to turn any pSB1C3 plasmid into a Bacillus subtilis compatible replicative plasmid.

Conclusions and perspectives

The start of our project has been complicated by the absence of available replicative plasmid for Bacillus subtilis in the registry. Even managing to obtained one was not the end of our quest since it appears to be too big for further sub-cloning purposes. In this context, obtaining the pSB_BS0K-Mini has been a great step forward for us as it allowed a fast progression of our project cloning steps.

Moreover, we are also very proud of the OriKan biobrick. There was no such part in the registry. Its capacity to simply functionalize any registry part for Bacillus subtilis (and likely some other gram positive strains) is invaluable for the ever growing numbers of iGEM projects based on these organisms. We therefore applied for the OriKan biobrick to be selected as “best composite part”.

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