Difference between revisions of "Part:BBa K3332085"

Line 11: Line 11:
 
Fig.1 Circuit
 
Fig.1 Circuit
  
This part can be used to test that if the tetR protein can repress the pLtetO-1 promoter after the pLtetO-1 promoter is characterized. With the expression of tetR, the bacteria can’t have fluorescence intensity.
+
This part can be used to test that if the pLtetO-1 promoter can work.
  
 
===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|none|500px|caption]]
 
[[File:Fig.2 pLtetO-1 E0420 pSB1C3 and pUC57(BBa K3332084) digested by EcoR I and Pst I.png|none|500px|caption]]
Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by ''EcoR'' I and ''Pst'' I
+
Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by ''EcoR'' I and ''Pst'' I(about 1026 bp)
 
[[File:2034 fig.3.png|none|500px|caption]]
 
[[File:2034 fig.3.png|none|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(about 3922 bp)
  
ps:E0420 is equal to B0034_E0020_B0015
+
Note:E0420 is equal to B0034_E0020_B0015
  
 
Protocol:
 
Protocol:

Revision as of 16:45, 26 October 2020


J23106-RBS-tetR-pLtetO-1-ECFP-terminator

A composite part to check the function of pLtetO-1 promoter.

With this part, the pLtetO-1 promoter can be tested by observing the fluorescence intensity.

Usage and Biology

caption

Fig.1 Circuit

This part can be used to test that if the pLtetO-1 promoter can work.

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(about 1026 bp)

caption

Fig.3 pLtetO-1_E0420_J23106_P0140_pSB1C3(BBa_K3332085) digested by Spe I and Pst I(about 3922 bp)

Note: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 OD 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.

Reference

[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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]