Team:Goettingen/Notebook/evolution September

Transformation of SP1 with aroA* and gat

03.09.18

The B. subtilis strain SP1 was transformed with correct plasmids that contain either aroA* and gat. After incubation over night at 37 °C transformants were isolated.

05.09.18

The positive clones were singularized and afterwards propagated on strach plates in order to check the amylase activity. The starch test was performed as described <a href="https://2018.igem.org/Team:Goettingen/Experiments">here</a>. Positive Strains were isolated for further analysis.

Resistence test of aroA* and gat Strains

07.09.18

Both constructs, SP1+aroA* as well as gat were propagated on CS-Glucose plates that contain 5 and 10 mM glyphosate. Each plate was incubated for 2 days at 37 °C.

The shown strain variants that have higher resistances than the wildtype strain SP1 are displayed in the table.

Plasmid Resistance
aroA*1 ✔
aroA*2 ✘
aroA*3 ✘
aroA*4 ✔
aroA*5 ✔
aroA*6 ✔
aroA*7 ✔
aroA*8 ✘
aroA*9 ✘
aroA*10 ✘
aroA*11 ✘
aroA*12 ✘
aroA*13 ✘
aroA*14 ✔
aroA*15 ✔
aroA*16 ✘
aroA*17 ✘
aroA*18 ✘
aroA*19 ✔

The shown strain variants of SP1 with gat that have increased resistance against glyphosate are shown in the following table.

Plasmid Resistance
gat7 ✘
gat9 ✔
gat12 ✔
gat14 ✔
gat18 ✘
gat21 ✘
gat23 ✔

The aroA* variants of SP1 are less resistant than the gat variants. The aroA* variants grew on 5 mM glyphosate, whereas the gat variants grew on 10 mM of the herbicide. Two positiv and resistant plasmids of each construct were sequenced additionally.

10.09.18

The variants aroA*10 and gat9 were checked via sequencing and the sequences are correct. Competent cells of the strains SP1+aroA*10 and gat9 were created. Additionally, those strains were cultivated in kryo stocks at -80 °C.

11.09.18

The competent strains SP1+aroA*10 and SP1+gat9 were transformed with cDNA of the Strain BP233 (ΔgltT). Additionally, a retransformation of both strains SP1+aroA*10 and SP1+gat9 was performed.

Functionality of aroA*

14.09.18

In order to find out whether the SP1+aroA* veriant that confers glyphosate resistance contains a functional EPSP synthase which is encoded by the aroA* gene. Therefor, the strains SP1 (WT), SP1+aroA* and SP1+aroA* ΔaroE were propagated on CS-Glucose plates.

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