Part:BBa_K3332085

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

Fig 1. J23106_B0031_tetR_B0015_pLtetO-1_B0034_ecfp_B0015

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

Characterization

The agarose gel electrophoresis images of target fragments are shown as below： [[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(about 1026 bp)

Fig 3. J23106_P0140_ pLtetO-1_E0420_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 100 ng/mL）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.

3.Add 4 mL 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 0.5 mL culture in each tube. All samples are centrifuged at 12000 rpm, 1 minute. Remove supernatant and add 500 µL 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:

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.

In the figure, there is no obvious difference in fluorescence intensity/OD600 between induction group and non-induction group of the negative control (J23100) and pLtetO-1_E0420. While the fluorescence intensity/OD600 of J23106_P0140_pLtetO-1_E0420 in induction group is higher than non-induction group obviously. That is to say, 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]