Team:Goettingen/Notebook/evolution September
Contents
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.
| 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 | ✔ |
| 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.
<img src="">
The picture shows the strains SP1, SP1+aroA* and two variants of the strain SP1+aroA*ΔaroE.
All of the strains grew on CS-glucose plates. That indicates that both of the SP1+aroA* ΔaroE variants contain a functional aroA*.