Team:Toulouse France/Description

iGEM Toulouse 2016

Project

How to help Lascaux ?

Introduction

The Lascaux cave, situated in the south-west of France, is one of the most important heritages we received from our ancestors. 18000 years ago, they performed there one of the most sensational collection of parietal frescoes and engravings. Decorated with organic and minerals pigments, it is home to bulls, aurochs and horses and testify the way of life of our ancestors.

Background - the historic of the cave

As soon as the cave was discovered in 1940, people came from the whole country and abroad to visit it. However, the carbon dioxide and the water vapour released by their breath started to disrupt the cave’s ecosystem. The damages caused by these air changements are at the origin of the first crisis that the Lascaux cave knew. Two kinds of contaminations were observed: the limestone formation on the walls, which was called the white disease, and the green algae growth, named the green disease.
Then, white mold and black stains appeared on the walls and the floor, and were characteristical of the second and the third crisis respectively. These contaminations flow from fungi and bacteria proliferation.

Treatment of the cave

Curing the cave was a priority. Consequently, treatments like biocid or antibiotics were tried. The success of these initiatives worked temporarily, but the cave ecosystem is a very unstable, and new microorganisms developed from the molecules previously used as treatment. Nowadays, the cave situation is steady thanks to the efforts of the cave curators, but scraping of contaminants is required on a daily basis.

Why did we choose this subject?

We wanted to choose a project that was personal and original. The Lascaux cave was self-evident to all of us. It is part of the French heritage, and was added to the UNESCO World Heritage Sites list. It has been threatened of destruction since decades now, and organizations from all around the world have gathered to solve the Lascaux cave crisis. Since mechanical and chemical solutions are insufficient, we reasoned that a biological solution should be try…and enrolled in the iGEM competition !

Introduction

The historic of the cave

Treatment of the cave

Why did we choose this subject?

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 BS 0K 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 BS 0K-Mini: new part BBa_K1937001

We reasoned that most of the pSB BS 0K 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 pSBBS0K plasmid. Position of the primers are indicated by the blue arrow on pSBBS0K (left part of the figure). The resulting PCR fragment is the blue pointed line. After digestion by NheI and self-ligation, the resulting pSBBS0K-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 BS 0K-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 casset. Position of the primers are indicated by the blue arrow on pSBBS0K (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 pSBBS0K 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 BS 0K-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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