Part:BBa_K3332088

pLtetO-1-RBS1-LacI-ssrAtag(mf-lon)-terminator-pTrc-2-RBS-ECFP-terminator

With this part, the pTrc-2 promoter can be tested by observing the fluorescence intensity.

Usage and Biology

This part can be used to test that if the LacI protein can repress the pTrc-2 promoter. With the expression of LacI, the bacteria have little fluorescence.

Fig 1. pLtetO-1_RBS1_LacI_terminator_pTrc-2_RBS(B0034)—ecfp-terminator

Characterization

We use pTrc-2 derivative_E0420_pUC57[BBa_K3332087],pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57 and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] to characterize pTrc-2 promoter and pTrc-2 derivative promoter. The agarose gel electrophoresis images are below:

Fig 2. pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by EcoR I and Pst I (about 1018 bp).

Fig 3. pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by Pst I (about 5086 bp).

Fig 4. pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by Pst I (about 5125 bp).

note: E0420 is equal to B0034_E0020_B0015.

Protocol:

1.Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution.

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

3.Add 4 mL of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 100 μL IPTG 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 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 5. Fluorescence intensity/OD for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form．

The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, pTrc2-derivative_E0420(ECFP) are used as the positive control group while pLtetO-1_LacI_pTrc2_E0420(ECFP) and pLtetO-1_LacI_pTrc2-derivative_E0420(ECFP) are both experimental groups.

We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved. The change of fluorescence intensity after induction of pLtetO-1-LacI-pTrc2-E0420(ECFP) group is larger than pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.

Sequence and Features

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]

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