Latest revision as of 03:26, 28 October 2020

mprA-protection coding3-mCherry reverse-emrr binding promoter reverse

Plasmid jqy is built as a biosensor of Salicylic acid and can also be used as a expression vector of mprA. Because it uses lacO, you need to use IPTG for induction. Its sequence includes a circuit of mprA and emrr Binidng Promoter. Mpra is a protein which will bind to emrr Binidng Promoter and cause expressing repression, this repressing function disappears after mprA is binding to Salicylic acid. So the understream gene will express if Salicylic acid exist. The plasmid also has a mCherry sequence as the understream gene of emrr Binding Promoter.

KSA_KOREA iGEM2020

How does the lignin biosensor work?

Our biosensor is composed of two main parts. The first part expresses mprA, which functions as a negative regulator, and the other part regulates the expression of the reporter gene. The mprA protein expressed by the constitutive promoter LacI binds to the emrR promoter and inhibits mScarlet expression. When Biosensor E. coli uptakes phenolic compounds (Fig. 1B), the mprA protein and the phenolic compound are bound, while the mprA protein is no longer bound to the emrR promoter. As a result, mScarlet becomes expressed.

FIGURE 1. SCHEMATIC DIAGRAM OF BIOSENSOR AND PHENOLIC COMPOUNDS DERIVED FROM LIGNIN

Evaluation of Biosensor

We first confirmed whether the previously reported biosensor (BBa_K2717017) was functional. Vanillin, which will be used as the phenolic compound, was purchased from the online market “Auction” and was prepared by dissolving it in anhydrous ethyl alcohol at a concentration of 1 M. Next, pBiosensor#1 plasmid containing the 1st biosensor part shown in Figure 2A was transformed into E. coli. Overnight cultured E. coli was diluted 1:100, transferred to 2 ml of new LB media, and treated with vanillin at the indicated concentration. E. coli was incubated for 8 hours at 37°C and 240 rpm in a shaking incubator. After each sample was transferred to a 2ml centrifuge tube and centrifuged at 13,000 rpm for 1 minute, the supernatant was removed. We resuspended pellets with 20ul of PBS and spotted them on LB agar plate. Images were acquired using camera attached to a fluorescence microscope. The samples treated with 4 mM vanillin showed stronger fluorescence than the negative control.

FIGURE 2. CONFIRMATION OF INCREASED EXPRESSION OF MSCARLET BY VANILLIN

Biosensor performance improvement

Previous studies have shown that CouP, an active transporter of phenolic compounds, is involved in vanillin uptake and further enhances the conversion of vanillic acid and catechol. So, we added CouP to improve the efficacy of the 1st biosensor. We designed the expression of CouP in two methods (constitutive or inducible expression) as shown in Fig. 3.

FIGURE 3. EXPRESSION OF COUP FOR EFFECTIVE TRANSFER OF PHENOLIC COMPOUNDS

1st Iteration First, to maintain the high level of CouP protein, we manufactured plasmid in a way that can be controlled by the T7 promoter and the lac operator. We prepared the transformation E. coli with the 2nd Biosensor plasmid in the same manner and dispensed IPTG according to concentration and incubated for 8 hours. Unlike our expectations, the sample in which IPTG was dispensed showed less fluorescence in contrast to that in which IPTG was not dispensed (Fig. 4).

FIGURE 4. DIFFERENCES IN BIOSENSOR FUNCTION ACCORDING TO COUP OVEREXPRESSION

2nd Iteration As the overexpression of CouP could affect the expression of the Reporter gene, we constructed a new plasmid DNA in which LacI promoter controls the CouP expression,. We followed the same procedure as the previous experiments, and observed brighter red fluorescence when CouP was present. After digitizing the pixel intensity of the fluorescence with the help of ImageJ, we found that the fluorescence was about 20% higher when CouP was present (Fig. 5).

FIGURE 5. IMPROVING THE PERFORMANCE OF BIOSENSORS BY CONSTITUTIVELY EXPRESSED COUP

Optimization Since we observed that the fluorescence varied on the type of E. coli strain during the cloning process of the plasmid DNA used in the biosensor, we tested which strain our biosensor functioned the best on. We transformed our 3rd biosensor plasmid in the three most commonly used strains (Mach1, BL21(DE3), DH5a). As can be seen from Figure 6, fluorescence was highest in BL21(DE3).

FIGURE 6. SELECTION OF HOST STRAINS TO MAXIMIZE MSCARLET FLUORESCENCE

Integration with Ligninatior

Finally, we confirmed that our biosensor works when we treated lignin that is degraded by laccase. Kraft lignin was degraded for 24 hours at 40°C by laccase in 100mM sodium acetate(pH 5) containing 1mM ABTS. The degraded lignin was treated in Biosensor E. coli, and the experiment was carried out in the same manner. As a result, the fluorescence was higher when treated with lignin degraded by laccase (Fig. 7).

FIGURE 7. DETECTION OF LIGNIN DEGRADATION PRODUCTS OF IMPROVED BIOSENSORS

4. Conclusion

Our biosensor showed 20% improved performance than the previously known biosensors. When CouP is overexpressed, it seems that mScarlet transcription and translation are inhibited. When CouP expression is regulated by a promoter such as mprA, the fluorescence value increases. E. coli BL21(DE3) is a strain deficient in ATP dependent serine protease, and it is thought that mScarlet protein is more stable than DH5ɑ or Mach1. Our biosensor was unable to discern fluorescence below 0.2 mM vanillin. Therefore, there is still a limit to detecting the phenolic compound in the environment. However, it could be applied to detect phenolic compounds in wastewater thrown away by paper companies.

Reference

Alvarez-Gonzalez, Neil Dixon, G. N. (2019, October 15). Genetically encoded biosensors for lignocellulose valorization. BMC.

Sequence and Features