Difference between revisions of "Part:BBa K3332040"
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<partinfo>BBa_K3332040 short</partinfo> | <partinfo>BBa_K3332040 short</partinfo> | ||
| − | A promoter derived from | + | A promoter derived from pTrc-2 promoter can be strongly repressed by LacI protein. pTrc-2 derivative promoter has three lac operators, which means LacI has a stronger inhibitory effect on it. |
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | The | + | The pTrc-2 promoter is used to express mf-lon and MazF in the absence of ATc so as to inhibit the growth of E.coli. It is part of the circut designed to prevent engineered E.coli in the detection instrument from escaping. |
| − | In this circuit, LacI can repress | + | In this circuit, LacI can repress pTrc-2 promoter and pTrc-2 derivative promoter，while tetR can repress pLtetO-1 promoter. When ATc exits, it can combine tetR, so that pLtetO-1 promoter can’t be repressed. Then LacI which is controlled by pLtetO-1 can repress pTrc-2 promoter and pTrc-2 derivative promoter. As a result, mf-lon and MazF can’t be expressed. |
| + | |||
| + | As a kind of bacterial toxin, MazF can cause the bacteria death. So there comes the conclusion that as long as the engineered E.coli are cultured in the environment with ATc, it won’t be killed by MazF, but when the E.coli escape from our testing instrument, the effect can be reversed, that is to say, the E.coli will be killed by MazF. In the same way, we can conclude that in the presence of IPTG, MazF can be expressed to cause bacterial death. | ||
| + | |||
<table><tr><th>[[File:T--XMU-CHINA--circuit--circuit.png|thumb|600px|Fig.1 Circuit.]]</th><th></table> | <table><tr><th>[[File:T--XMU-CHINA--circuit--circuit.png|thumb|600px|Fig.1 Circuit.]]</th><th></table> | ||
===Characterization=== | ===Characterization=== | ||
| + | We use pTrc-2 derivative_E0420_pUC57[BBa_K3332087],pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] to characterize pTrc-2 derivative promoter. | ||
| + | |||
The agarose gel electrophoresis images are below: | The agarose gel electrophoresis images are below: | ||
| − | + | ||
| − | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|300px|Fig. | + | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|300px|Fig.2 pTrc-2 derivative_E0420_pUC57[BBa_K3332087] digested by <i>EcoR</i> I and <i>Pst</i> I.]]</th><th></table> |
| − | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332088.png|thumb|300px|Fig. | + | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332088.png|thumb|300px|Fig.3 pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] digested by <i>Pst</i> I.]]</th><th></table> |
| − | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332089.png|thumb|300px|Fig. | + | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332089.png|thumb|300px|Fig.4 pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] digested by <i>Pst</i> I. note: E0420 is equal to B0034_E0020_B0015]]</th><th></table> |
===Protocol=== | ===Protocol=== | ||
| − | 1. Preparation of stock solution | + | 1. Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution |
| − | + | ||
2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h. | 2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h. | ||
| + | |||
3.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm. | 3.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm. | ||
| − | 4.Add 100μL IPTG 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. | + | |
| + | 4.Add 100μL IPTG 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. | 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. | 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: | Here is the result: | ||
| − | <table><tr><th>[[File:T--XMU-CHINA--figure 14.png|thumb|600px|Fig. | + | <table><tr><th>[[File:T--XMU-CHINA--figure 14.png|thumb|600px|Fig.5 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.]]</th><th></table> |
| − | The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure | + | |
| − | <table><tr><th>[[File:T--XMU-CHINA- | + | 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 the pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that the LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter. | ||
| + | |||
| + | <table><tr><th>[[File:T--XMU-CHINA-yingguang.png|thumb|600px|Fig.6 In each group，the EP tube on the left is non-induction group while the one on the right is induction group.]]</th><th></table> | ||
From this figure, the induction effect can be seen more intuitively. | From this figure, the induction effect can be seen more intuitively. | ||
===Sequence And Features=== | ===Sequence And Features=== | ||
Revision as of 11:34, 26 October 2020
pTrc-2 derivative
A promoter derived from pTrc-2 promoter can be strongly repressed by LacI protein. pTrc-2 derivative promoter has three lac operators, which means LacI has a stronger inhibitory effect on it.
Usage and Biology
The pTrc-2 promoter is used to express mf-lon and MazF in the absence of ATc so as to inhibit the growth of E.coli. It is part of the circut designed to prevent engineered E.coli in the detection instrument from escaping. In this circuit, LacI can repress pTrc-2 promoter and pTrc-2 derivative promoter，while tetR can repress pLtetO-1 promoter. When ATc exits, it can combine tetR, so that pLtetO-1 promoter can’t be repressed. Then LacI which is controlled by pLtetO-1 can repress pTrc-2 promoter and pTrc-2 derivative promoter. As a result, mf-lon and MazF can’t be expressed.
As a kind of bacterial toxin, MazF can cause the bacteria death. So there comes the conclusion that as long as the engineered E.coli are cultured in the environment with ATc, it won’t be killed by MazF, but when the E.coli escape from our testing instrument, the effect can be reversed, that is to say, the E.coli will be killed by MazF. In the same way, we can conclude that in the presence of IPTG, MazF can be expressed to cause bacterial death.
Characterization
We use pTrc-2 derivative_E0420_pUC57[BBa_K3332087],pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57[BBa_K3332088] and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] to characterize pTrc-2 derivative promoter.
The agarose gel electrophoresis images are below:
Protocol
1. Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution
2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
3.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
4.Add 100μL IPTG 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:
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 the pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that the LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.
From this figure, the induction effect can be seen more intuitively.
Sequence And Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 37
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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