TITLE: Testing safety mechanism
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In order to test the efficiency of the kill switch mechanism,  we plan on performing the following experiments:</div><div class = "text-block"><span style = "font-weight:bold;">Test amino acid autotrophy </span><span style = "font-weight:bold;font-style:italic;">in vitro</span></div><div class = "text-block">An overnight culture will be used to inoculate a test culture. The knock out strains will be grown in a medium containing the test amino acid and then transfered to a minimal medium lacking the specific amino acid. Samples of 0.5 ml will be removed every 30 min to test the OD. </div><div class = "text-block"><span style = "font-weight:bold;">Testing solanine dependency </span><span style = "font-weight:bold;font-style:italic;">in vitro</span></div><div class = "text-block">A test culture will be inoculated from the overnight culture. The cells will be grown in LB supplemented with solanine (CONCCCCCC). The cells will be collected, washed and resuspended in LB lacking solanine. Samples of 0.5 ml will be removed every 30 min in order to test OD. </div><div class = "text-block"><span style = "font-weight:bold;">Testing dependency in soil</span></div><div class = "text-block">The GFP-mutant strains (amino acid auxotrophs and solanine dependent mutants) will be inoculated at the root of the potato plants. The potato plants will be grown and described as above.</div></div>

[STEPS]
?. [Test safety mechanisms in the lab]
Plate mutants on minimal media supplemented with the auxotrophy amino acid or solanine.
?. [Test safety mechanisms in the lab]
Pick one colony and inoculate . Grow B. mycoides
5 mL
30 33
?. [Test safety mechanisms in the lab]
The next day inoculate a working culture (  media + 1% overnight culture)
30 mL
?. [Test safety mechanisms in the lab]
Grow at   until OD600nm is 0.5
30 33
?. [Test safety mechanisms in the lab]
Collect cells by centrifugation
Centrifuge: 4000 34, 10 min, 4 10
?. [Test safety mechanisms in the lab]
Wash 5 times with growth media (without amino acid)
This step may need ajustment. The purpose is to eliminate the dependency molecule from the growth medium
?. [Test safety mechanisms in the lab]
Resuspend in   minimal media.
30 mL
?. [Test safety mechanisms in the lab]
Divide culture in 2 x  cultures.Supplement one of them with the required amino acid.
15 mL
?. [Test safety mechanisms in the lab]
Incubate both samples at
30 33
?. [Test safety mechanisms in the lab]
Every  take a  sample and check OD600
0.5 mL
?. [Test safety mechanisms in the lab]
The OD of the sample + amino acid will increase exponentiallyThe other one will remain 0.5
?. [Test safety mechanism in soil]
Grow potato plants in a 40 cm diameter pot.
?. [Test safety mechanism in soil]
Inoculate   of a GFP-labeled B. mycoides (auxotroph) culture in exponential growing phase (OD600nm=0.5)For a negative control use GFP-labeld B. mycoides.
5 mL
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?. [Test safety mechanism in soil]
Take soil samples of control and mutants from:- as close to the root as possible- 10 cm distance fro m root- 20 cm distance from root- 30 cm distance from root - 40 cm distance from root - 50 cm distance  from root