Difference between revisions of "Part:BBa K2825002"

Revision as of 10:47, 27 October 2020

PcaU inducible promoter with high sensitivity

Inducible promoter device for protocatechuic acid (PCA). The PcaU transcription factor represses its own transcription as well as a target transcript when PCA is not present by binding to the operator region. When PCA is present, PcaU releases the region and transcription of the target transcript occurs.

This version of PcaU is derived from evolutionary engineering courtesy of Jha et. al. See design section for details

Usage and Biology

PcaU is an IclR transcription factor originally obtained from a PCA catabolism operon in Acinetobacter baylyi. (Jha et al., 2018). PcaU was then optimized for sensitivity and specificity in response to PCA in Pseudomonas putida through directed evolution.

Characterization

PcaU has been inserted into the pGLO plasmid, in which it expresses GFP when induced by PCA. Fluorescent intensity scales hyperbolically with PCA concentration. Above 10mM concentration, PCA was toxic to BL21 E. coli containing the PcaU reporter. We used a logarithmic scale in the chart below to clearly indicate the relationship between PCA concentration and fluorescence.

The lower range of PcaU sensitivity is in single micromolar concentrations. When measuring PCA concentrations this dilute in a multi well plate, wells on the edge of the plate show increased error. For accurate results, fill outer wells with water and use inner wells only. The figure below used 32 wells in the center of a 48 well plate (n=8). Washing wells in PBS will also reduce error. We used a logarithmic scale in the chart below to clearly indicate the relationship between PCA concentration and fluorescence.

PcaU is functional in DH5a E. coli, but is more sensitive in BL21 DE3 E. coli. We used a linear scale in the chart below to show the equal uninduced fluorescence of these cells.

For parts submission, we mutated an EcoR1 site in the noncoding region from GAATTC to GAAATC. The chart below shows that the mutation did not affect sensitivity. We used a linear scale in the chart below to show the equal uninduced fluorescence of these cells.

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
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 171

References

1. Jha, R. K., Kern, T. L., Fox, D. T., & M. Strauss, C. E. (2014). Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry. Nucleic Acids Research, 42(12), 8150–8160. http://doi.org/10.1093/nar/gku444

@@ Line 42: / Line 42: @@
-==Improve the p3B5C==
-[[Part:BBa K3395015|PcauAM-p3b5b]] was obtained by point mutation of [[Part:BBa K2825002|PcauAM-p3b5c]],iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part [[Part:BBa K2825002|PcauAM-p3b5c]].
-==Characterization==
-The [[Part:BBa K3395015|PcauAM-p3b5b]] system is constructed with two parts, upstream activator, and downstream reporter.[[Part:BBa K3395015|PcauAM-p3b5b]] was obtained by point mutation of [[Part:BBa K2825002|PcauAM-p3b5c]],iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part [[Part:BBa K2825002|PcauAM-p3b5c]]. The target of this system is to test the sensitivity of the PcauAM activator and p3b5b promoter combination. The upstream sequence is composed of  [[Part:BBa_K3395008|pLacIQ promoter]], [[Part:BBa_K3395006|PcauAM]] and [[Part:BBa_K3395014|tonB terminator]] . The upstream sequence will express PcauAM protein, which is not stable, and denature itself. The downstream sequence is composed of [[Part:BBa_K3395009|p3b5b promoter]], [[Part:BBa_K3395007|eYFP reporter]] and L3S3P21(terminator). This downstream system will present high expression when PcauAM protein is generated. We use eYFP to show the sensitivity of this combination.
-===Experment===
-We constructed the promoter into the low copy p15A plasmid with YFP downstream its regulatory protein PcauAM. We transformed pET28b_p3B5B vector into <i>E .coli</i> BL21 for testing.
-[[File:T--RDFZ-China--pcauAM p3B5B.png|400px|thumb|center|Biosensor circuit of pcauam_p3B5B]]
-===Experimenting===
-Activity test:
-Sensor activity of PcaU-pPCA, PcaUAM-p3B5B, PcaUAM-p3B5C system under different emission light.
-[[File:T--RDFZ-China--sensor activity test.png|400px|thumb|center|Result of activity test]]
-The data in the right of figure are the tested data of PcaUAM-YFP under emission light 525nm.
-When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM-p3b5c are both about 3000. And when PCA is added, the YFP protein expression of the two sensing systems has increased to different degrees, and the value of F/A is increased when reflected in the image. The data shows both p3b5b and p3b5c promoter can be controlled by the pcauAM activator. And under the horizontal comparison between p3b5b and p3b5c promoter, p3b5b promoter have much higher sensitivity than p3b5c promoter. However, consider our downstream system will be TPH1 sequence, we don't want the control system that is too sensitive, because too much TPH1 express might cause the 5-HTP symptom.
-Comparing the leakage and sensitivity, we found that PcauAM expressor-p3b5b downstream promoter are the best sensor system considering the standard of our project. It has the relatively lower leakage comparing with PcaU-pCPA system, also with relately low sensitivity comparing to PcaUAM-p3B5C promoter.