Difference between revisions of "Part:BBa K3332089"
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<partinfo>BBa_K3332089 short</partinfo> | <partinfo>BBa_K3332089 short</partinfo> | ||
| − | + | With this part, the LacI protein can be tested by observing the fluorescence intensity. | |
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | + | This part can be used to test that if the LacI protein can repress the Ptrc-2 derivative promoter after the Ptrc-2 derivative promoter is characterized. With the expression of LacI, the bacteria can’t have fluorescence intensity. | |
| − | < | + | <table><tr><th>[[File:T--XMU-CHINA--circuit4.png.png|thumb|600px|Fig.1 Circuit.]]</th><th></table> |
| − | < | + | ===Characterization=== |
| + | The agarose gel electrophoresis images are below: | ||
| + | <table><tr><th>[[File:T--XMU-CHINA-BBa K3332086.png|thumb|300px|Fig.2 pTrc-2_E0420_pSB1C3[BBa_K3332086] digested by <i>Xba</i> I and <i>Pst</i> I.]]</th><th></table> | ||
| + | <table><tr><th>[[File:T--XMU-CHINA--BBa K3332087.png|thumb|300px|Fig.3 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.4 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.5 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=== | ||
| + | 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: | ||
| + | <table><tr><th>[[File:T--XMU-CHINA--figure 14.png|thumb|600px|Fig.6 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 below, pTrc2-derivative are used as the negative control group, the pTrc2-derivative are used as the positive control group while the pLtetO-1-LacI-pTrc2-E0420 (tetR) and pLtetO-1-LacI-pTrc2-derivative-E0420(tetR) are both experience group. We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved and the pLtetO-1-LacI-pTrc2-E0420 (tetR) group the difference of fluorescence intensity is smaller than the pLtetO-1-LacI-pTrc2-derivative-E0420(tetR) group so we can confirm that the LacI has a weak inhibitory effect on pTrc-2 promoter. That’s why after adding IPTG, the fluorescence intensity of pLtetO-1-LacI-pTrc2-E0420 (tetR) group increases faster. | ||
| + | <table><tr><th>[[File:T--XMU-CHINA--pLtetO-1-LacI-pTrc2-E0420.png|thumb|600px|Fig.7 In each group，the EP tube on the left is without induction while the one on the right is with induction.]]</th><th></table> | ||
| + | From this figure, the induction effect can be seen more intuitively. | ||
| + | ===Sequence and Features=== | ||
<partinfo>BBa_K3332089 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3332089 SequenceAndFeatures</partinfo> | ||
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<partinfo>BBa_K3332089 parameters</partinfo> | <partinfo>BBa_K3332089 parameters</partinfo> | ||
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| + | ===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 | ||
Revision as of 04:21, 25 October 2020
pLtetO-1-RBS1-LacI-ssrAtag(mf-lon)-terminator-pTrc-2 derivative-RBS-ECFP-terminator
With this part, the LacI protein can be tested by observing the fluorescence intensity.
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