Team:Goettingen/Notebook/evolution May

02.05.18

The strains BP233, BP235 and the WT strain 168 were propagated to 3 ml LB medium and inoculated for 4 h at 37°C and 220 rpm. They were harvested in a 2 ml reaction tube by centrifugation at 13000 rpm for one minute. The supernatant was discared and the cells were washed in 1x C-Salts. Afterwards, they were resuspended in 300 µl 1x C-Salts. The OD600 was determined.

Strain OD600
168 9.8
BP233 6.1
BP235 7.6

The cells were then struck out on cs-glucose agar plates with variating glyphosate concentrations. 168 was propagated on plates containing 0 mM and 10 mM glyphosate, while BP233 was additionally propagated on plates with the glyphosate concentrations of 30 mM and 40 mM.BP235 was additonally propagated on plates containing glyphosate concentrations of 50 mM and 60 mM. All agar plates were then inoculated at 37 °C for 2 days.

The strains 168, BP233 and BP236 were propagated to 3 ml LB medium and inoculated for 4 h at 37°C and 220 rpm. They were harvested in a 2 ml reaction tube by centrifugation at 13000 rpm for one minute. The supernatant was discared and the cells were washed in 1x C-Salts. Afterwards, they were resuspended in 300 µl 1x C-Salts. The OD600 was determined.

Strain OD600
168 10.74
BP233 8.37
BP236 7.71

100 µl of solutions were propagated on CS-Glucose agar plates containing 0 mM glyphosate as well as plates containing 10 mM glyphosate. These plates were inoculated at 37 °C for 2 days.

03.05.18

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.

           <img src="T--Goettingen--notebook-may-030518.jpg">

Growth of the strains 168 and BP233 in absence of glyphosate and in presence of 10 mM glyphosate.

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!

04.05.18

The growth of the suppressor mutants of the strains 168, BP233 and BP235 on 10 mM and 0 mM glyphosate CS-glucose agar plates was documented.

<img src="T--Goettingen--168_0mMGS_040518png.png">

168 colonies were picked from the 10 mM glyphosate plate and steaked out on CS-glucose 10 mM glyphosate plates.

A growth test of 168, BP233 and BP237 on CS-Glucose agar plates containing 0 mM and 10 mM glyphosate.

Growth experiments

07.05.18

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. 

                   <img src="T--Goettingen--notebook-may-sp1_070518.png">

                   <img src="T--Goettingen--notebook-may-168_070518.png">

Growth curves of the wild type SP1 and the 168 mutant, auxotrophic for tryptophan.

The mutants of 168 grown on CS-glucose 10 mM glyphosate plates were inoculated at 37°C in liquid LB medium and used for cryo cultures (1-17).

08.05.18

BP233 colonies grown on the 40 mM glyphosate medium were inoculated on 40 mM glyphosate CS-glucose medium.

BP235 colonies from 50 mM glyphosate medium were inoculated on 40 mM CS-glucose medium

Cryo cultures of mutant strains were made named igem18-34 and cultures 6-8 were inoculated again.

14.05.18

Over night cultures were prepared for cryo cultures.

Preparing of over night cultures for cDNA extraction.

15.05.18

Cryo cultures of mutant strains were made named igem35-44.

The cells of the over night culture were harvested and the cDNA was isolated according to the <a href="https://2018.igem.org/Team:Goettingen/Experiments" target="_blank" >script</a>

Identification of novel mutations in B. subtilis 168

16.05.18

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.

           <img src="T--Goettingen--Notebook_suppressor_160518.png">
Mutant names DNA concentration in [ng/µL]
iGEM18 15.2
iGEM19 44.5
iGEM20 57.4
iGEM21 48.3
iGEM22 45.3
iGEM23 54.3
iGEM24 44.7
iGEM25 31.9
iGEM26 29.7
iGEM27 80.7
iGEM28 58.8
iGEM29 51.9
iGEM30 57.7
iGEM31 /
iGEM32 56.1
iGEM33 62.2
iGEM34 42.5

Amplification of the gltT gene

17.05.18

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

Component Volume in [µL]
10x Taq buffer 5
dNTPs (12.5 mM) 2
fwd Primer: iGEM2018_15 2
rev Primer: iGEM2018_16 2
B. subtilis chromosomal DNA 1
Dream Taq polymerase 0.25
sterile H2O 37.75
Total 50
Results

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

                   <img src="T--Goettingen--Notebook_gltTamplification1_170518.png">

                   <img src="T--Goettingen--Notebook_gltTamplification2_170518.png">

Amplified gltT gene from 168, iGEM19/20.

Amplified gltT gene from iGEM30/32/33/34.

           <img src="T--Goettingen--Notebook_gltTamplification3_170518.png">

Amplified gltT gene from 168, iGEM18/21/22/23/24/25/26/27/28/29.

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

New Primers for gltT and sequencing

23.05.18

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
                   <img src="T--Goettingen--Notebook_gltTamplification_new1_230518.png">

                   <img src="T--Goettingen--Notebook_gltTamplification_new2_230518.png">

Amplified gltT gene from iGEM18–iGEM29.

Amplified gltT gene from iGEM30–iGEM34.

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.

gltT sequence Mutation Consequence
Wild type — —
iGEM18 Δ669A Truncation
iGEM19 Δ669A Truncation
iGEM20 C211T Truncation
iGEM22 duplication 839–847 Insertion
iGEM23 Δ65T Truncation
iGEM24 deletion 898–923 Truncation
iGEM25 duplication 839–847 Insertion
iGEM27 A388T Truncation
iGEM29 duplication 839–847 Insertion
iGEM30 Δ996&nash;1090 Truncation
iGEM32 Δ669A Truncation
iGEM33 duplication 839–847 Insertion

25.05.18

The wildtype strain 168 as well as the strains BP233 and BP236 were inoculated in 3 ml LB medium for 2-4h. 1.5 ml of each culture were harvested via centrifugation for 1 min at 13000 rpm. The supernatent was discarded and the cells were resuspended in 1 ml 1x C-salts. The were centrifuged again and the supernatent was discarded. The cells were resuspended in 400 µl 1x C-salts. 100 µl of each strain were plated on CS-glucose agar plates containing no glyphosate and plates containing 10 mM glyphosate.

Agar plates from the 25.05.18

28.05.18

<img src="T--Goettingen--Agarplatten_280518.png">

31.05.18

Preparation of CS-glucose (without trp) and CS-glucose containing 50 mM glyphosate.

SP1 was inoculated in LB medium over night.