Difference between revisions of "Part:BBa K2789018"

 
 
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<partinfo>BBa_K2789018 short</partinfo>
 
<partinfo>BBa_K2789018 short</partinfo>
  
 
MazF is a toxin coding gene that encodes toxin proteins that express the degradation of mRNA and inhibit the translation process, thus killing bacteria.MazE is an antitoxin encoding gene, which can encode and MazF encoding the antitoxin to bind to each other and make it lose its virulence. However, the antitoxin protein is unstable and easy to be degraded by serine protease.
 
MazF is a toxin coding gene that encodes toxin proteins that express the degradation of mRNA and inhibit the translation process, thus killing bacteria.MazE is an antitoxin encoding gene, which can encode and MazF encoding the antitoxin to bind to each other and make it lose its virulence. However, the antitoxin protein is unstable and easy to be degraded by serine protease.
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===Material:===
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====(1) DNA element :====
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1. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG<br/>
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2. MazF/MazE: MazF is a toxin-encoding gene that encodes toxin proteins that express degradation of mRNA to inhibit translation, thereby killing bacteria. MazE is an anti-toxin-encoding gene that encodes an anti-toxin protein that can bind to the MazF-encoded toxin to make it lose toxicity, but the anti-toxin protein is unstable and easily degraded by serine proteases.<br/>
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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.<br/>
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4. TetO : TetO could be inhibited by tetR)<br/>
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====(2) Plasmids:====
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1. Prefix + lac operator + MazF + riboswitch + MazE + suffix<br/>
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===Experiments:===
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1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.<br/>
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2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium<br/>
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3. Cultivate the medium for 2 hours in 37°C<br/>
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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<br/>
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5. Cultivate at 37 degrees, shaker 200 rpm culture<br/>
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6. The OD600 was measured for 6h and 12h, respectively, to determine the number of viable bacteria.<br/>
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===Results:===
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  <img src="https://static.igem.org/mediawiki/2018/d/d3/T--WHU-China--parts-Parts_rg5.png">
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Because theophylline itself may be harmful to cells, as a substance that turns off the riboswitch, we first made a gradient dilution and found the mildest range for the bacteria.<br/>
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Although we used a lower concentration of theophylline in this pathway, we finally found that this pathway did not work.<br/>
  
  

Latest revision as of 13:36, 17 October 2018

In the absence of ligand, the expression of toxin inhibits the reproduction of bacteria so as to pre

MazF is a toxin coding gene that encodes toxin proteins that express the degradation of mRNA and inhibit the translation process, thus killing bacteria.MazE is an antitoxin encoding gene, which can encode and MazF encoding the antitoxin to bind to each other and make it lose its virulence. However, the antitoxin protein is unstable and easy to be degraded by serine protease.


Material:

(1) DNA element :

1. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG
2. MazF/MazE: MazF is a toxin-encoding gene that encodes toxin proteins that express degradation of mRNA to inhibit translation, thereby killing bacteria. MazE is an anti-toxin-encoding gene that encodes an anti-toxin protein that can bind to the MazF-encoded toxin to make it lose toxicity, but the anti-toxin protein is unstable and easily degraded by serine proteases.
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 + MazF + riboswitch + MazE + 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:

Because theophylline itself may be harmful to cells, as a substance that turns off the riboswitch, we first made a gradient dilution and found the mildest range for the bacteria.
Although we used a lower concentration of theophylline in this pathway, we finally found that this pathway did not work.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]