Difference between revisions of "Team:Goettingen/Notebook/evolution May"

Strains BP233, BP235 and 168 (WT) were incubated in 4 ml LB medium for 4 h at 37°C and 220 rpm. 2 ml of the culture was harvested, and the cells were washed in 1xC-salts and then resuspended in 300 µl 1xC-salts. BP233 and BP235 were plated on CS-glucose with and without 10 mM glyphosate.

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As seen in this figure, both strains grow well on CS-glucose without glyphosate. Strain BP233 also forms a lawn on plates with 10mM glyphosate, while the wildtype does not form a lawn, but single colonies, which could be suppressor mutants. This leads to the hypothesis that GltT, which is a glutamate transporter, transports glyphosate into the cell. The strain BP235 was not stable, so it was constructed again!

B. subtilis 168 and B. subtilis SP1 were transferred into CS-Glucose medium containing increasing glyphosate concentrations to an OD₆₀₀ of 0.1. OD measurement was carried out every 2 hours until the cells reached an OD₆₀₀ of ∼2. The cells were harvested by centrifugation for 1 min at 13000 rpm and stored at -20°C.

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For the identification of novel mutations in the gltT transporter, the wild type strain was grown on 40 mM glyphosate. Emerging mutations were further analysed by isolation of the complete DNA and amplification of the transporter via PCR. The genomic DNA was isolated with the Kit “peqGOLD Bacterial DNA Kit” from peqlab. The resulting sequences were then screened for novel mutations.

For the identification of novel mutations in the gltT transporter, the wild type strain was grown on 40 mM glyphosate. Emerging mutations were further analysed by isolation of the chromosomal DNA and amplification of the transporter via PCR. The genomic DNA was isolated with the Kit “peqGOLD Bacterial DNA Kit” from peqlab.

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We amplified the gltT gene from B. subtilis with a PCR run with the following recipe.

Results

In the following figures are the results of the PCR shown.

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→ iGEM21 and iGEM26 did not show any PCR products. Therefore, no sequence could be further analyzed.

The PCR from 17.05.18 was repeated with new primers (iGEM2018_19 and iGEM2018_20) that amplify also the ends of the gene gltT. The protocol was the same as before.

Results

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The amplified gltT fragments were purified and sent to sequencing with primers iGEM2018_29 and iGEM2018_30. The following table provides an overview of mutations in gltT from glyphosate adapted mutants.