Part:BBa_K2789015
In the absence of ligand, the expression of toxin inhibits the reproduction of bacteria so as to pre
ParD is a toxin coding gene that encodes toxins that express toxins that inhibit DNA helicase activity, thereby inhibiting bacterial reproduction. ParE is an antitoxin coding gene that encodes and parD encodes the antitoxin proteins that bind to each other to render them toxic, but the antitoxin proteins are unstable and easily degraded by serine protease. Antitoxins can prevent the effects of endogenous expression of toxins, but in the case of large amounts of toxin expression, antitoxins are difficult to antagonise toxins, which can lead to the death of bacteria Used to prevent horizontal gene transfer of bacteria
Material:
(1) DNA element :
1. parD/parE: parD is a toxin-encoding gene that encodes a toxin that inhibits DNA helicase activity, thereby inhibiting bacterial growth. parE is an anti-toxin-encoding gene that encodes an anti-toxin protein that can bind the parD-encoded toxin to make it lose toxicity, but the anti-toxin protein is unstable and easily degraded by serine proteases. 2. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG 3. Theophylline ribose switch: When theophylline is present, the theophylline and the riboswitch are combined, so that the nearby RBS cannot be further used, so that the translation cannot be completed. 4. TetO : TetO could be inhibited by tetR)
(2) Plasmids:
1. Prefix + lac operator + parD+ riboswitch +parE + suffix
Experiments:
1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight. 2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium 3. Cultivate the medium for 2 hours in 37°C 4. Take 30ul of the inoculum to the theophylline concentration of 0, 0.1, 0.2, 0.4, 0.8, 1.6 (units g / L) and 0, 0.5, 1, 2, 4, 6, 8 (units g / L) 10 ml of LB liquid medium 5. Cultivate at 37 degrees, shaker 200 rpm culture 6. The OD600 was measured for 6h and 12h, respectively, to determine the number of viable bacteria.
Results:
We performed this pathway validation with a lower theophylline concentration. We can drew the results that this pathway can work and the best effect of theophylline concentration is 0.2g/L.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 150
Illegal NgoMIV site found at 658 - 1000COMPATIBLE WITH RFC[1000]
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