Part:BBa_I715039
pLac-RBS-RFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 781
Illegal AgeI site found at 893 - 1000COMPATIBLE WITH RFC[1000]
Measured by BIT-China 2019
The function of BBa_I715039 is confirmed by our 2019 team.
Experimental steps:
The fluorescence intensity of cells is usually characterized by relative fluorescence intensity, that is, the average fluorescence intensity per unit of OD600 cells.The specific measurement process is as follows:
1.After fermentation, 500ul samples were centrifuged at room temperature at 12000rpm for 1min.
2.Discard the supernatant and remove the possible residual liquid as far as possible.
3.Bacterial precipitates were resuspended with 500ul10mM PBS solution.
4.The suspension was diluted with PBS solution to OD600 =1.0;
5.The 200ul diluted bacterial solution was added into a transparent 96-well culture plate and a black 96-well plate, respectively.
6.The OD600 value of bacteria in the transparent pore plate was determined by microplate reader.The fluorescence intensity of bacteria in black pore plate was measured.
7.The final relative fluorescence intensity of the cells was determined as the measured fluorescence intensity divided by the measured OD600.
We successfully constructed pUC19-mRFP plasmid and pUC19 plasmid expressing plasmid and measured the activity of these parts quantitatively. In this time,we introduced both of mRFP expressing plasmid and control group expressing plasmid into the same strain, difference in mRFP expression was observed dependent on cultivation time. The culture of the strain with pUC19-mRFP at 37℃ showed about 70 folds higher fluorescence intensity than control group, While the culture of the strain with pUC19-mRFP and control group at 37℃ at Initial stage of fermentation showed almost the same fluorescence intensity. No inducer was added during the entire fermentation process.This result indicates that the lac promoter is leaked without the addition of IPTG induction.
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