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_B0034_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：

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 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:

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]