Part:BBa_K861171:Experience

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Methods

Design of the promoter Pcar which is activated by glucose

Promoter Pcar , glucose biosensor plasmid, is derived from constitutive promoter (BBa_J23119) by adding a CRP binding site upstream the promoter which has several base pairs overlapping with polymerase binding site. The sequence was synthesized with restriction enzyme cutting site for EcoRI and XbaI at the 5' terminal and SpeI at 3' terminal. The sequence of promoter Pcar has cohesive terminus at both ends, so it is very convenient for us to construct the plasmid for functional detection.The sequence of Pcar is as followed:

Construction of plasmid for direct regulation pathway

In this experiment, RFP reported the function of the indirect regulation pathway.

BBa_K861179: BBa_I13507, an mRFP generator with RBS and terminator was embedded downstream the constitutive promoter BBa_J23119

BBa_K861176: BBa_I13507 was embedded downstream the artificial promoter Pcar.

BBa_K861178: a constitutive expressed CRP(J23116+K861161) was assembled with K861176.

All new composite parts mentioned above were transformed to competent cells of Escherichia coli str. DH5a. All positive clones are validated using PCR, restriction enzyme digestion and DNA sequencing.

Functional detection

The same methods with that of the indirect regulation pathway were used to confirm that the promoter worked as expected. For details, please click Here.

Results

Construction of the plasmid for functional detection

The sizes of the Promoter Pcar and J23119 were less than 100 bp and proved to be correct by the agarose gel electrophoresis . Restriction Digestion of the plasmid BBa_I13507 only have one lad on the agarose gel, it told that the plasmid was digested well. After transformation, competent cells were cultured on agar plate with 50 μg/L of ampicillin. Both red and white bacterial colonies emerged on one plate. The red ones were the correct clones revealing promoter embedded successfully, while the white ones were negative . The red clones were picked out and cultured in LB medium for plasmid extraction. Purified plasmids were digested with XbaI and PstI for confirmation. The bands of 2000bp and 1000bp showed that the promoter had been embedded successfully. At last, the plasmids we acquired were sent for sequencing, results show no mutation exist.

Cell culture fluorescence measurement

The correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader. All fluorescence was normalized with absorbance at 600 nm.The results represented the fluorescence of every cell.
The fluorescence of K861179 was about 10000 Relative Light Units. It did not vary with concentrations of glucose. However we found a positive correlation between the fluorescence of K861176 and concentration of glucose. At a glucose concentration lower than 4mM, the fluorescence was very low, but at high concentration of glucose like 100mM, the fluorescence was much less than that of K861179.

Capturing of the fluorescent image

Fluorescent images indicated that all the cells were growing normally, because the size and morphology were both the same with cells in LB medium.the fluorescence of the cells in the images show the same discipline with results from the fluorescence measurement experiments. Fluorescence of K861179 was very strong but it didn't change with the glucose concentration. On the contrary, fluorescence of K861176 was relatively weak but increased with concentration of glucose.

Fluorescent analysis of cyto-imaging

The results of FANCY is showed as bellow, single cell was recognized from fluorescence images and fluorescence intensity was caculated.In the table, datas show that RFP expression was activated in high glucose concentration, which conforms well with results above.For more information about FANCY,click Here.

 

Discussion

The promoter Pcar is a promoter designed for the Eschaerichia coli which is derived from a constitutive promoter BBa_J23119. Pcar includes the CRP-binding site and the RNA polymerase-binding site which overlap several base pairs. Therefore, because of the steric hindrance between CRP and RNA polymerase, gene downstream of the promoter will be repressed at high concentration of CRP. In the cells, low glucose concentration results in increasing activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to bind tightly to the specific DNA site in the promoter and repress the gene downstream. On the contrary, high glucose concentration will result in the expression of the promoter.

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