Engineering

Design

To biosynthesize indigo, we selected gene EcTnaA and gene MaFMO based on literature. Although EcTnaA (tryptophanase) is native to Escherichia coli, its low endogenous expression required exogenous introduction. EcTnaA cleaves L-tryptophan to indole, which is then oxidized by MaFMO to 3-hydroxyindole. Spontaneous condensation of 3-hydroxyindole forms indigo with water release.^[1]

We cloned these genes into the pRSFDuet-1 vector existing in the lab under a T7 promoter for IPTG-induced expression, testing whether this dual-enzyme system could produce indigo in E.coli.

Build

All cloning procedures were performed in SnapGene. The two genes were assembled into the plasmid backbone by restriction–ligation. EcTnaA was inserted with NcoI and NotI sites, whereas MaFMO was inserted with BglII and XhoI sites. After sequential digestion and ligation, two genes were seamlessly inserted into the vector.

Figure 1: History record of pRSFDuet-1-EcTnaA-MaFMO plasmid

Test

The assembled plasmid was transformed into Escherichia coli BL21(DE3). Cells were first cultured in glucose-containing fermentation medium and induced with 0, 0.2, 0.4, or 0.6 mM IPTG. After 24 h and 48 h, concentrated glucose and tryptophan solutions were fed to the cultures.^[2]Both induction and fermentation were carried out at 30 °C. No visible color change was observed at 48 h (Figure 2).

Figure 2: The state of the bacterial liquid after 48 hours

Protein expression was subsequently assessed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) (Figure 3) and Western blot. In the Figures, 0, 0.2, 0.4, and 0.6 denote IPTG induction concentrations of 0 mM, 0.2 mM, 0.4 mM, and 0.6 mM, respectively.

Figure 3: SDS-PAGE electrophoresis result image

Figure 4: Western blot result image of protein EcTnaA

Western blot showed that EcTnaA was not expressed as expected; the observed band was at the wrong molecular weight.

Figure 5: Western blot result image of the protein MaFMO

Western blot showed that MaFMO was expressed as expected; the observed band was at the right molecular weight（56.0KDa）.

Learn

After consulting relevant materials, we learned that indole is toxic to cells, and the function of the EcTnaA protein is to convert L-tryptophan into indole. If the induced expression temperature is too high, it will cause the EcTnaA protein to fold incorrectly and form inclusion bodies. Our experiment adopted an induction condition of 30°C. Therefore, we believe that the excessively high induction temperature led to the incorrect expression of EcTnaA protein, thus failing to produce our target product, indigo.

In addition, we have also learned that the glucose in the glucose fixation medium used for bacterial culture will undergo the Maillard reaction with tryptophan at high temperatures, thus being unable to be correctly utilized by bacteria. Moreover, the structure of tryptophan will also change at high temperatures.

Redesign

We adjusted the temperature and time for induction and enzyme activity, and used LB medium to cultivate the bacteria. We added 0.2mM IPTG and 0.4mM IPTG to induce expression, and re-conducted Western Blot experiments to verify the expression of the protein EcTnaA. After confirming the correct expression of the protein, we added glucose concentrate and tryptophan concentrate to the culture medium and re-fermented and cultured to produce indigo.

Rebuild

We added 0.2mM and 0.4mM concentrations of IPTG to the culture medium respectively to induce expression, and incubated at 16°C and 190rpm for 16 hours. Then, glucose concentrate and tryptophan concentrate are added, and the bacteria are cultured through two-stage fermentation.

Retest

After induction at 16°C, blue bubbles were clearly visible in the culture medium (Figure 6).

Figure 6: 0.2 mM IPTG (left), 0.4 mM IPTG (right), 16 °C induction, 16 h

We conducted a Western Blot experiment, and the results indicated that the EcTnaA protein was correctly folded and expressed (Figure 7).

Figure 7: Western blot results of EcTnaA protein

After adding glucose and tryptophan, the blue color significantly darkened after 24 hours of culture at 30°C and 200rpm. Moreover, blue particles can be observed in the culture medium (Figure 8).

Figure 8: 0.2 mM IPTG (left), 0.4 mM IPTG (right), 30 °C fermentation, 24 h

After adding glucose and tryptophan and continuing to culture for 24 hours, it can be seen that the number of blue particles increases and the color of the culture medium darkens (Figure 9).

Figure 9: 0.2 mM IPTG (left), 0.4 mM IPTG (right), 30 °C fermentation, 48 h

Relearn

From failure to success in indigo fermentation, we accumulated the experience of failure, adjusted the induction temperature and fermentation conditions, and achieved engineering success. During the experiment, we noticed that tryptophan and glucose would react at high temperatures, and the structure of tryptophan would denature at high temperatures. Therefore, we did not use glucose medium but adopted the method of preparing glucose concentrate and tryptophan concentrate to provide the raw materials for fermentation.

At the same time, we flexibly adjusted the induction temperature and fermentation temperature of IPTG, hoping to first allowE.coli to express the corresponding enzyme at 16°C and achieve the best activity of the enzyme at 30°C, so as to better ferment indigo.

Furthermore, by setting different IPTG induction concentrations, we found that the protein expression was the best and the indigo yield was the highest under the induction of 0.2mM concentration IPTG.

Design

Since our ultimate goal is to produce indigo under blue light irradiation, we need to modify the promoter of the previously constructed pRSFDuet-1-EcTnaA-MaFMO plasmid, as the target genes in this plasmid are driven by the T7 promoter and T7 terminator. We decided to use homologous recombination and inverse PCR methods to replace the T7 promoter and T7 terminator with the pPhlF promoter and L3S3P11 terminator.

Build

We designed inverse PCR primers to linearize the plasmid(Figure 10), aiming to remove the first T7 promoter before the MCS1 region. Meanwhile, we used designed homologous recombination primers to amplify the pPhlF promoter from the cloning vector pUC57-MaFMO-EcTnaA by PCR.

Figure 10: Results of plasmid inverse PCR. Lanes 1, 2, 4, and 5 show bands consistent with the expected size of 5113bp.

We used Vazyme's ClonExpress MultiS One Step Cloning Kit (C113-01) to perform homologous recombination ligation between the promoter fragment and the linearized plasmid fragment. To enhance experimental rigor, we set up a blank control group where reaction products without the homologous recombinase Exnase MultiS were transformed into E.coli DH5α for culture.

Test

Our culture results did not match expectations, as the number of colonies on the negative control plate was not significantly fewer than that on the experimental group plate(Figure 11). We therefore adopted colony PCR to further verify whether the recombinant vector was successfully introduced into E.coli DH5α.

Figure 11: Growth of bacteria on plates from homologous recombination experiment. Left(a): Experimental group; Right(b): Negative control group.

Figure 12: Results of colony PCR

The colony PCR results showed that our homologous recombination reaction failed(Figure 12), and we were unable to successfully ligate the promoter with the linearized plasmid.

Learn

We began to review the experiment and finally speculated that primer dimerization was severe. Additionally, with the promoter sequence being only 51bp, the amplified fragment was similar in length to the primer dimer fragments, making it difficult to determine whether the promoter was successfully amplified. Therefore, we used Oligo software for theoretical verification(Figure 13) and designed experiments for practical validation(Figure 14). We performed PCR with only forward and reverse primers without adding the plasmid template to observe if a sequence around 100bp would appear.

Figure 13: Oligo analysis showing severe self-dimerization of the reverse primer

Figure 14: Results of experimental verification. Template√ indicates the addition of plasmid template; Template× indicates no plasmid template added.

Based on the above verification, our method of modifying the promoter via homologous recombination failed because the promoter sequence was too short, and our primer design range was also limited. Although this experiment failed, it ruled out a less feasible method for our subsequent systematic promoter modification.

Redesign

After the failure of the homologous recombination method, we consulted our experimental instructor and decided to amplify the entire target gene fragment pPhlF-MaFMO-EcTnaA-L3S3P11 terminator, then insert it into the vector pRSFDuet-1 using restriction enzyme digestion and ligation.

Rebuild

We operated in SnapGene, using the restriction enzymes NcoI and NotI to insert the target gene into the vector.

Figure 15: History record of constructing the pRSFDuet-1-MaFMO-EcTnaA-L3S3P11 vector on SnapGene

Retest

We introduced the constructed expression vector into E.coli DH5α. Colonies showing positive results in colony PCR were subjected to Sanger sequencing. Sequence alignment confirmed that our target gene was successfully ligated into the vector without mutations.

When our promoter pPhlF is bound to the PhlF protein, it will be unable to drive the expression of the target gene. However, when the blue light light-controlled plasmid is not introduced and the PhlF protein cannot be expressed in the bacteria, the promoter pPhlF will not be inhibited and will normally drive the expression of downstream genes. So we introduced the constructed recombinant plasmid into E.coli BL21 (DE3) for fermentation. After 24 hours of culture, we clearly saw indigo pigment particles produced in the culture medium (Figure 16).

Figure 16: Results of plasmid introduction into E.coli BL21 (DE3) for 24h after promoter modification (a, b)

Relearn

This experimental cycle taught us that when the promoter sequence is too short, making PCR amplification difficult, and when sequences such as the T7 promoter are difficult to remove from the vector, we can consider amplifying the entire segment containing the promoter and using restriction enzyme digestion and ligation to connect it to the vector. Moreover, it is not necessary to completely remove the T7 promoter, Lac operator, and Lac sequences.