Part:BBa_K4591009
T500-RFP-lox66-Hpall-lox71-XylSmut-tetO-sfGFP-T500
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Usage and Biology
In order to observe the decomposition of PET by engineered bacteria, our team designed this plasmid. The plasmid mainly consists of RFP, lox66&lox71, PHpaII, Xylsmut, tetO and sfGFP genes. It is mainly used to detect and report the presence of PA and TPA, and visually monitor the expression through the GFP.When the TPA is detected by the Xylsmut, the downstream genes will be activated and transcribed——like the strain will express green fluorescence.
Fig.1. A kind of simple XylSmut-based fluorometric biosensors which can detect the TPA.
What’s more, considering that in future product applications, the strain may not necessarily be used to degrade PET immediately after delivery, we designed the flipping system to cope with different use situations. During the strain is put into use to decompose the PET, we can regulate the flipping sequence to detect the presence of TPA and activate the expression of GFP. In other situations, flipping expresses the RFP and plays an alternative role. For example, when applied in a factory, in order to reduce adverse effects during fermentation production, the engineered bacteria express antibacterial products before turning over, and then turn over to express downstream degradation modules until they are put into use. Or in the field, the strain produces substances that increase fertility into the soil before the sequence is flipped. In turn, the engineered bacteria could have more purposes.
Fig.2. Shows how the flip module work
Basic components of the circuit
RFP：It is a strong reporter that has RFP expression that can be detected with a fluorescent plate reader or visualized by the unaided eye. 37°C was the optimal growth temperature for E. coli expressing the RFP and the expression/detection of the RFP increased over time with 48 hours showing the most robust red color. [From Part:BBa_E1010]
Fig.3. Picture of visible RFP expression at different incubation times [From Part:BBa_E1010]
XylS can bind benzoic acid and various derivatives, but it cannot recognize PA and TPA.[Part:BBa_K4591002] So by directed evolution of a promiscuoustranscription factor, XylS from Pseudomonas putida, Jiawei Li and Mario Roque Huanca Nina successfully created twonovel variants, XylS-K38R-L224Q and XylS-W88C-L224Q, thatare able to bind PA and TPA.[1]
Such XylS mutants can be used to build PA and TPA biosensors that detect the presence of TPA and initiate the expression of the corresponding modules. This component is able to regulate the expression of downstream sfGFP gene to report the breakdown of TPA and the Pm promoter.
Fig.4. A kind of simple XylSmut-based fluorometric biosensors which can detect the TPA
lox71&lox66：n this project, the lox sequence is used for specific segment flipping, and in bacteria, the sequence is complete and invertible.The lox sequences, lox71 and lox66, have 5 bp on the 5 and 3 ends changed, respectively. DNA segment flanked by lox71 and lox66 marks the point which the enzyme Cre will excise. This Part is expected to express GFP when the lox sites are excised and RFP when they are not. According to the iGEM11_Tokyo_Tech team, the Cre-mediated recombination of this BioBrick had been studied and proved to be working.In in vivo assay, arabinose induced strain which has Cre-expressing plasmid(PBAD/araC-Cre, BBa_I718008) was expressing GFP, while negative control which doesn't have Cre plasmid was expressing Red florescence. It means that DNA recombination did happen by Cre recombinase. [Part:BBa_K649202]
PHpaII：The PHpaII promoter[2], is a strong constitutive promoter that is commonly used for expression in gram-positive bacteria.This promoter has a similar expression intensity to P43 and a better affinity for binding sites of RNA polymerase or other transcriptional activators. It helps to increase the transcription rate of downstream DNA to messenger RNA and stimulates counterclockwise RNA synthesis.[3,4,5]It is used with lox sequences for flipping specific sequences in our case.
TetO：DNA regulatory element comprising an array of seven TetO regions, and the TetR protein binds to TetO to inhibits gene expression.
GFP: Green fluorescent protein, modified from the original GFP, is used to test the effectiveness of recombinant enzymes and bistable systems. Green fluorescent protein (GFP) was discovered in 1961 as a byproduct of the extraction of aequorin from the Auquorea victoria jellyfish. [6]
Characterization
In the flip module we constructed a much simpler sequence of flips, as shown below:
Fig.5. A much simpler sequence of flips for testing
We wanted the flip module to have RFP characterization before cre recombinase expression and sfGFP characterization after importing cre recombinase.
We first introduced the flipping module (pMA5) into BL21, shook the bacteria overnight at 37℃ 200rpm, centrifuged the bacterial solution, discarded the supernatant, and observed it under UV light.
Then transform and introduce plasmid with cre recombinase, add 0.1% xylose to induce cre recombinase gene expression to flip recombinase target sites, shake the bacteria overnight, take the bacterial solution and centrifuge, discard the supernatant, add water to resuspend the bacteria and observe under UV light.
Fig.6. The right one was the engineered bacteria without the Xylose and the sfGFP was expressed.
The left one with xylose added shows that the RFP was expressed.
The result shows that, the cre recombinase successfully reverse the direction of the T7 promoter and express the different protein.
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