Difference between revisions of "Team:Toulouse France/Notebook"

Dry lab:

Now that the subject is defined, and that the bibliography is done, let’s prepare our constructions! What we have done:

• selected and adapted genes
• designed primers
• ordered gBlocks
• ordered the material (enzymes, kit…)necessary for the project

Wet lab:

Having a good control of the basic experiments, as enzymatic digestion or transformation for example, is important to ensure the lab work during the summer. We have done PCR, digestion, ligation, and transformation for clonings during a week, being supervised by PhD students.

Dry lab:

In parallel, we finished ordering gBlocks from IDT, and we refined the gene design.

Wet lab:

First of all, we had to digest the backbone pSB1C3, which have to received each of our parts. Thus we obtained high quantity of pSB1C3 plasmids with RFP insert due to cultures, and we purified and digested it with the enzymes EcoRI and PstI. As the gBlocks were received, we did PCR to amplify them and create a stock. Most of the PCR worked, but not all of them. Thus, we start trying to clone the good amplicons in the backbone pSB1C3, but it fail because of a lack of genetic material.
We asked to iGEM Munich 2012 to send us four shuttle backbones, that can be used in E. Coli as well as in B. subtilis. We amplified them and stocked them. In parallel, we have done petri dishes with several antibiotic resistances to have it in advance in stock.
In 2014, the iGEM Toulouse team worked on fungi, and constructed strains able to secrete antifungals. We used these strains in order to: have a positive control when our bacteria will be functional, test the efficiency of the antifungals, and find the best temperature, medium and technique. We also determined what was the more efficient between using the supernatant, the bacteria, or a mix of both. All these tests were performed on a fungi mat.

Dry lab:

We decided to create a device to confine our modified bacteria. We designed it using Blender.

Wet lab:

The transformation of E. coli with the construction of a NagA inducible promoter in pSB1C3 worked, it was the first big step! However, transformation with our antifungals and toxin genes did not work. As the cloning with pNagA is validated, we started trying to clone this insert in the shuttle plasmid pSB0K-P from 2012 iGEM Munich team, and to transform it in E. coli.
Some gBlocks were still in process to be amplified by PCR. We tried different programs, by modifying the elongation time or the hybridization temperature.
The genes (sdp and skf) for our predation module should be cloned by the Gibson method. The clonings did not work.

Dry lab:

As the PCR of gBlocks do not work very well, we designed other primers to improve the compatibility between the template and the primers.
For the modelling, we designed the prey-predator model to have a simulation of the activity of our modified B. subtilis.

Wet lab:

We continued trying to clone our insert pNagA in the shuttle plasmid pSB0K-P, using different strategies. Indeed, we tried to digest the DNA fragments with different enzymes. Nevertheless this plasmid is quite big (>9 kbp) and thus it is harder to clone than a smallest plasmid. As a consequence, we thought about a strategy to reduce the size of this plasmid.
The cloning of some gBlocks in pSB1C3 worked! They are an antifungals part and toxin/antitoxin genes. We started to clone it in pSB0K-P and to transform it in E. coli.
We also had problem with the Gibson cloning.

Dry lab:

Moreover, in order to reduce the size of our replicative plasmid pSB0K-P, we designed primers that were supposed to amplify both the origin of replication and the antibiotic resistance gene for B. subtilis and E. coli.

Wet lab:

The bacteria we are modifying is supposed to improve the predation capacity of B. subtilis. In order to determine if our bacteria is more efficient than the wild type bacteria, we have done predation tests by using the strain WT 168. The bacteria that our B. subtilis is supposed to limit the growth is Pseudomonas fluorescens SBW25.
We have done PCR on the big pSB0K-P plasmid to reduce its size, from 9,3 kbp to 5,7 kbp, hoping the cloning will become easier. We started to clone our gBlocks pNagA, mazF and AfA in this plasmid.
Problems with the cloning in pSB1C3 of our predation genes were still present.
Dry lab:

We designed new primers to optimize the Gibson cloning of our predation genes.

Wet lab:

The iGEM Imperial College of London asked us to collaborate with them. We had to determine the optimum growth conditions (pH and temperature) of Bacillus subtilis and Pseudomonas fluorescens. We have done experiments realizing cultures in different conditions during the all day, and we measured the OD every hour. Due to these data we determined the doubling time of each conditions, and we selected the best one.
Now that the small version of the plasmid pSB0K-P is circularized, we transformed it in Bacillus subtilis. The cloning of our three inserts in the pSB0K-Mini worked, and we also succeed in transforming B. subtilis with these constructions.

Wet lab:

The problems with the Gibson cloning are still present, but the origin of the disturbance is unknown.
We started to test our promoter pNagA in B. subtilis. It was supposed to be inducible in presence of NagA, and then give off RFP. In parallel, we tried to test the efficiency of our antifungal operon. It was useful to determine if our B. subtilis is able to affect the growth of other bacteria.

Wet lab:

The plasmid pSB0K-Mini can be replicated in E. coli, but the antibiotic resistance functional in this bacterium is the ampicillin. One of our objective was to modify it to remplace the ampicillin resistance gene by a chloramphenicol resistance. By amplifying by PCR pSB0K-P to select only the kanamycine gene and the replication origin in B. subtilis, and by ligating it with pSB1C3, we should obtain a plasmid replicative in E. coli and in B. subtilis. In addition, the antibiotic resistance genes would be the one expected: chloramphenicol for E. coli, and kanamycine for B. subtilis.

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