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.
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===Usage and Biology:===
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[[File:Fig1.circuit .png|center|500px|caption]]
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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.
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===Characterization:===
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The agarose gel electrophoresis images are below：
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[[File:Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I.png|center|500px|caption]]
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Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I
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[[File:2034 fig.3.png|center|500px|caption]]
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Fig.3 pLtetO-1_E0420_J23106_P0140_pSB1C3(BBa_K3332085) digested by Spe I and Pst I
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ps：E0420 is equal to B0034_E0020_B0015
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Protocol:
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1. Preparation of stock solution
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Dissolve ATc in absolute alcohol to make 1000× stock solution（the work concentration is 100ng/mL）
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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4.Add 200 µL ATc stock solution into the induction group when OD600 increased to 0.6.
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5.Induce for 6 hours and the condition is the same as before.
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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.
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7.Measure the fluorescence intensity（ECFP）and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD value of each group.
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Here is the result:
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[[File:2034 fig4.tif|center|500px|caption]]
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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.
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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.
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===Source===
===Source===
Latest revision as of 01:20, 25 October 2020
pLtetO-1
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Design Notes
None.
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
