Difference between revisions of "Part:BBa K3332034:Design"
(→‎Design Notes)
Line 9: Line 9:
 
pLtetO-1 promoter is a promoter from E. coli that can be repressed by the protein tetR(BBa_C0040 or BBa_K3332039).When there is anhydrotetracycline （ATc）, the repression will be inhibited.  
 
pLtetO-1 promoter is a promoter from E. coli that can be repressed by the protein tetR(BBa_C0040 or BBa_K3332039).When there is anhydrotetracycline （ATc）, the repression will be inhibited.  
  
−
Usage and Biology:
+
===Usage and Biology:===
 
[[File:Fig1.circuit .png|center|500px|caption]]
 
[[File:Fig1.circuit .png|center|500px|caption]]
 
pLtetO-1 promoter was used to express LacI(ssrAtag(mf-lon)) to construct a monostable kill switch. Without ATc, tetR is expressed to repress pLtetO-1, then E. coli will be killed.
 
pLtetO-1 promoter was used to express LacI(ssrAtag(mf-lon)) to construct a monostable kill switch. Without ATc, tetR is expressed to repress pLtetO-1, then E. coli will be killed.
  
−
Characterization:
+
===Characterization:===
 
The agarose gel electrophoresis images are below：
 
The agarose gel electrophoresis images are below：
 
[[File:Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I.png|center|500px|caption]]
 
[[File:Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I.png|center|500px|caption]]
Line 19: Line 19:
 
[[File:2034 fig.3.png|center|500px|caption]]
 
[[File:2034 fig.3.png|center|500px|caption]]
 
Fig.3 pLtetO-1_E0420_J23106_P0140_pSB1C3(BBa_K3332085) digested by Spe I and Pst I
 
Fig.3 pLtetO-1_E0420_J23106_P0140_pSB1C3(BBa_K3332085) digested by Spe I and Pst I
 +
 
ps：E0420 is equal to B0034_E0020_B0015
 
ps：E0420 is equal to B0034_E0020_B0015
  
Line 34: Line 35:
 
Fig.4 Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
 
Fig.4 Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
 
We discovered that 100ng/mL ATc can inhibit the repression of tetR on pLtetO-1, then turn on the expression of downstream gene of pLtetO-1.
 
We discovered that 100ng/mL ATc can inhibit the repression of tetR on pLtetO-1, then turn on the expression of downstream gene of pLtetO-1.
 +
 
===Source===
 
===Source===
  

Revision as of 01:11, 25 October 2020


pLtetO-1


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]


Design Notes

pLtetO-1 promoter is a promoter from E. coli that can be repressed by the protein tetR(BBa_C0040 or BBa_K3332039).When there is anhydrotetracycline （ATc）, the repression will be inhibited.

Usage and Biology:

caption

pLtetO-1 promoter was used to express LacI(ssrAtag(mf-lon)) to construct a monostable kill switch. Without ATc, tetR is expressed to repress pLtetO-1, then E. coli will be killed.

Characterization:

The agarose gel electrophoresis images are below：

caption

Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I

caption

Fig.3 pLtetO-1_E0420_J23106_P0140_pSB1C3(BBa_K3332085) digested by Spe I and Pst I

ps：E0420 is equal to B0034_E0020_B0015

Protocol: 1. Preparation of stock solution Dissolve ATc in absolute alcohol to make 1000× stock solution（the work concentration is 100ng/mL） 2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h. 3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm. 4.Add 200 µL ATc stock solution into the induction group when OD600 increased to 0.6. 5.Induce for 6 hours and the condition is the same as before. 6.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500µL sterile PBS to resuspend. 7.Measure the fluorescence intensity（ECFP）and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD value of each group. Here is the result:

caption

Fig.4 Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction. We discovered that 100ng/mL ATc can inhibit the repression of tetR on pLtetO-1, then turn on the expression of downstream gene of pLtetO-1.

Source

Ordered from Genscript company.

References

[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979