Indole Degradation Pathway

Cycle 1 Substitute the Chassis Strain

Design

Considering template availability and time constraints, we adopted a strategy of assembling functional components obtained through gene synthesis separately. Given plasmid incompatibility, we opted for pUC57 and pUSP as the framework for this segment.

Build

The plasmids were introduced into E.coli Top10 using the heat shock method.

Test

The piGEM23_04 pathway for indole degradation remained genetically stable in E.coli DH5α. Subsequently, we conducted plasmid extraction and introduced it into the Top10 strain for expression and functional testing. The Top10 strain was cultured in LB medium for the subsequent detection of indigo and isatin production, but it proved to be intolerant and unable to grow.

Learn

Through literature review, we discovered that both EPI400 and Top10 are competent E.coli strains commonly used for gene cloning, efficient expression, and protein production enhancement. However, they exhibit differences in crucial aspects. The EPI400 strain was developed to significantly reduce the copy number of many common vectors, making it easier to clone unstable DNA sequences.

Cycle 2 Substitute the Growth Medium

Design

We substituted the host strain for the indole degradation pathway with E.coli EPI400 and proceeded with subsequent protein expression and degradation capability tests after introducing piGEM23_04.

Build

Using EPI400 as the host strain, we employed the heat shock method to introduce piGEM23_04 into E.coli, followed by quantifying protein expression through SDS-PAGE. We measured the absorbance peak at 571nm, indicative of the rose indole product resulting from the reaction of Kovac's reagent with indole, to reflect the amount of indole degradation. Additionally, we measured the production of indigo and isatin using a UV-Visible spectrophotometer.

Test

In the SDS-PAGE analysis, we successfully detected the expression of ycnE and FMO enzymes from piGEM23_04 in the bacterial cells. In addition, a decrease in indole concentration in the culture broth could be detected. Subsequently, we cultured EPI400 in LB medium and conducted measurements for the subsequent production of indigo and isatin. However, the presence of endogenous indole in LB medium made it challenging to maintain consistent substrate levels for accurate testing. Furthermore, the high absorbance at the characteristic absorption peak of isatin in LB affected the accuracy of the isatin standard curve, leading to imprecise detection of isatin production.

Learn

Through literature research, we identified that LB medium is a nutrient-rich culture medium primarily composed of abundant elements, trace elements, and organic compounds. The presence of tryptophan in LB medium serves as a critical substrate for E.coli to produce indole. Therefore, we need to switch to a medium devoid of tryptophan. M9 medium, also known as a minimal medium, has a well-defined composition. It consists of glucose as the carbon source, ammonium chloride as the nitrogen source, and other components to maintain osmotic pressure. M9 medium does not contain tryptophan, making it suitable for the subsequent testing of E.coli.

Cycle 3 Successful Isatin Detection

Design

Reverting to the use of M9 medium for cultivating EPI400 with imported piGEM23_04, we will proceed to conduct subsequent tests for protein expression and degradation capability.

Build

Employing M9 medium once again for culturing EPI400 with imported piGEM23_04, we will employ a UV-Visible spectrophotometer to measure the production of indigo and isatin.

Test

By switching to M9 medium, we have eliminated interference caused by endogenous indole production by E.coli and mitigated interference at the characteristic absorption peak of isatin in LB medium. Consequently, we successfully measured the production of indigo and isatin.

Learn

We have observed that the enzyme ycnE exhibits high efficiency in its function. To enhance the indole degradation rate, we aim to further improve the degradation efficiency of piGEM23_04.

Cycle 4 Single-point Mutation to Enhance Degradation Efficiency

Design

Considering directed evolution of the enzyme ycnE, we will employ virtual mutagenesis techniques to predict the structures of all mutations for each residue in ycnE. Subsequently, we will conduct molecular docking of all single-point mutations with the substrate indole. After screening, we will identify mutations with higher affinity than the wild-type ycnE and further confirm them using molecular dynamics simulations.

Build

Utilizing homologous recombination, we will construct seven variants (F75S, F75T, F75A, E54S, E54A, E54T, H65W) based on three mutation hotspots, and test the isatin production for the final screened variants.

Test

Through comparative analysis, we observed a significant enhancement in the functionality of ycnE for four mutated variants (F75S, E54S, E54A, and E54T) compared to the wild-type ycnE. Specifically, the activity of these variants increased by approximately 1.03 to 1.92 times compared to the wild type, indicating a significant enhancement in functionality through targeted mutation of specific amino acid residues in ycnE.

Learn

Employing directed evolution and virtual mutagenesis can significantly enhance enzyme activity.

Vector Construction

Cycle 1 Preliminary Design of the Plasmid

Design

Considering template availability and time constraints, we adopted a strategy of assembling functional components obtained through gene synthesis separately. Recognizing plasmid incompatibility, we chose pUC57 and pUSP as the scaffolds for this segment.

Build

Enzymatic cleavage at the BsmbI site, followed by homologous recombination using Gibson assembly, resulted in plasmid assembly. The assembled plasmid was then transformed into E.coli DH5α for amplification.

Test

In this regard, the indole degradation pathway piGEM23_04 and the formaldehyde degradation pathway piGEM23_03 can be stably inherited in E.coli DH5α. However, the pathways for nicotine, Benzo[a]pyrene, butyric acid, hydrogen sulfide, and ammonia degradation cannot be stably inherited in E.coli DH5α; the bacteria are intolerant to these pathways.

Learn

Actively analyzing the reasons and seeking assistance from the PI, we learned that the J23100 promoter is a constitutive promoter with overly strong expression, causing E.coli to be intolerant. Upon further research, we discovered that inducible promoters exhibit milder expression. Consequently, we decided to replace the promoters of several pathways with the lactose promoter for a gentler expression.

Cycle 2 Final Design of the Plasmid

Design

After replacing the J23100 promoter with a lactose promoter, we redesigned the plasmid vector construction.

Build

Enzymatic cleavage was performed at the BsmbI site, followed by homologous recombination using the Gibson assembly method to assemble the plasmid. Subsequently, the assembled plasmid was transformed intoE.coli DH5α for amplification.

Test

The degradation pathways for nicotine, Benzo[a]pyrene, butyric acid, hydrogen sulfide, and ammonia are all stably inherited in E.coliDH5α, and the bacteria are tolerant.

Learn

We successfully synthesized all genes for the degradation of target compounds.