ENGINEERING

We hope to verify the pathway from L-Tyr to naringenin in E. coli, so we designed three kinds of plasmids respectively contain matB-matC genes, TAL-4CL genes and CHS-CHI genes in order to synthesis naringenin using L-Tyr as ingredients. And simultaneously we want to verify the efficiency of cre-loxp principle in E. coli.In addition, we use B. subtilis to verify the pathway from naringenin to luteolin.

Objective: To prepare for the transformation of pCDFDuet-TAL-4CL into E. coli and the expression of naringenin.

Practice: pACYCDuet-matB-matC+pETDuet-CHS-CHI were transformed into E. coli. And then E. coli with these two plasmids was then made into chemically competent state.

Conclusion: Before transforming the two targeted plasmids into E. coli, we transformed pACYCDuet and pETDuet empty plasmids into E. coli and coated it on plates with homologous antibiotics. After a few times of failure,we saw bacterial colony on the plates, indicating the success of the transformation process. This showed the low probability of successfully transforming two or more plasmids into one bacteria simultaneously, while it is known to all that transform one plasmid at a time costs a longer time. Therefore, we chose to firstly electrotransform two plasmids into E. coli and secondly chemically transform the one plasmid left into the same E. coli.

Objective: To construct a engineering bacteria to achieve the goal of synthesizing naringenin. Thus we can verify the pathway from L-Tyr to naringenin.

Practice: pCDFDuet-TAL-4CL was successfully transformed into E. coli with the former two plasmids.

Conclusion: We can achieve the goal of simultaneously transforming numerous plasmids into the same E. coli even when we use two different methods of transformation.And eventually the sequencing analysis proofed that the transformation was a success.

Objective: To check whether there is expected enzymes existing or not.

Practice: We used wild-type BL21 strain (under different temperatures of 16℃, 25℃, and 37℃) as the Control group and BL21 strain with three plasmids under different temperatures (16℃, 25℃, and 37℃) as the Experiment group to do SDS-PAGE measurement.

Conclusion: We successfully obtained the enzymes that we want for producing naringenin no matter under what temperature conditions because there exist stripes in all Experiment groups and there do not exist any stripes in all Control groups. These results showed that the transformation and expression processes were successful.

Objective: To check whether there is naringenin produced or not, which is to check whether the first half of the pathway is successful or not.

Practice: We put the engineering bacteria under different temperature conditions, which are 16℃, 25℃, and 37℃ (37℃ is the temperature at which the bacteria are cultured, 25℃ is the room temperature, and 16℃ is the temperature we calculated according to the principle of similar span) for reaction and used HPLC to measure the concentrations of the naringenin under different conditions and compared them. By the way, we used wild-type BL21 strain as the Control group.

Conclusion: No matter what temperature is used for the reaction, there always exists naringenin in the Experiment group, while there is not any naringenin in the Control group, indicating that the first half of the pathway is feasible.

Objective: To construct a plasmid that can achieve the pathway from naringenin to luteolin.

Design plasmid: According to the pathway from naringenin to luteolin, we use gene FNS to turn naringenin into apigenin, a kind of intermediate product, and then we use gene F3'H to turn apigenin into luteolin. We chose the pBE2R vector to hold these gene fragments, and apart from the gene fragments, we added a terminator onto the pBE2R vector.

Practice: We used the principle of PCR to duplicate fragments of RBS1-FNS1, RBS2-F3'H-TT, vector-pBE2R, and a terminator. And then we used the method of homologous recombination to connect these fragments and the vector into a whole plasmid.

Conclusion: We initially tried to construct a plasmid whose structure is p43promoter-FNS-terminator-p43promoter-F3'H-terminator, but because there exist many similar sequences which cause the homology to be poor, we could not use the method of homologous recombination to successfully connect these fragments. Therefore, we deleted a promoter and a terminator that are located between genes FNS and F3'H, and then the homologous recombination succeeded.

Objective: To obtain the engineering bacteria that can produce luteolin.

Practice: We made BS168 strain into chemically competent state. And used the method of fast transformation to transform pBE2R-FNS-F3’H into BS168 strain.

Objective: To see whether there are targeted enzymes in engineering bacteria.

Practice: We got the protein samples from engineering bacteria with different plasmids. The one with pBE2R-FNS-F3'H is the Experiment group, while the wild-type is the Control group. We set temperature ladders of 16℃, 25℃, and 37℃ (37℃ is the temperature at which the bacteria are cultured, 25℃ is the room temperature, and 16℃ is the temperature we calculated according to the principle of similar span) for both the Experiment and Control groups. Then we conducted SDS-PAGE measurement to see if we got our targeted protein.

Conclusion: Our results from the SDS-PAGE measurement showed that temperatures don't have an evident influence on the expression process. And more importantly, we successfully obtained our targeted enzymes.

Objective: To further verify the presence of target enzymes in engineered bacteria. (Bands obtained from SDS-PAGE may be miscellaneous; antibody specificity is used to confirm that the obtained bands are target bands.)

Experimental Procedure: Take the protein gel samples obtained after SDS-PAGE: engineered bacteria containing the pBE2R-FNS-F3'H plasmid serve as the experimental group, and wild-type BS168 strain as the control group. Perform membrane transfer on the protein samples, add primary antibody and secondary antibody after blocking, then capture images for observation.

Conclusion: For the engineered bacteria with the pBE2R-FNS-F3'H plasmid, clear target bands are visible in the images; for the wild-type BS168 strain, no bands are observed. This confirms that the bands obtained from SDS-PAGE are the target bands.

Objective: To check whether there is luteolin in the reaction system so that we can know whether the second half of the pathway is feasible or not.

Practice: We put the engineering bacteria under different temperature conditions, which are 16℃, 25℃, and 37℃ (37℃ is the temperature at which the bacteria are cultured, 25℃ is the room temperature, and 16℃ is the temperature we calculated according to the principle of similar span) for reaction and used HPLC to measure the concentrations of luteolin under different conditions and compared them. By the way, we used wild-type BS168 bacteria as the Control group.

Conclusion: In our first attempt, we did not see any sign of luteolin’s occurrence. We reckon that the probable reasons may be as follows:

1. The plasmid we constructed was wrong, so the enzymes cannot perform their expected function.
2. It is hard for the ingredient naringenin to be transported into the engineering bacteria, which means that there lacks naringenin in the bacteria cell, so the enzyme cannot function well.

Because our sequencing result was very successful, we can rule out the influence of the first problem. To figure out whether it is the second reason influencing, we decided to construct an extracorporal reaction system. We explored different concentration conditions of naringenin, which are 0.1mM and 1mM. What’s more, we explored different reaction time conditions, which are 1h, 2h, 3h, 4h, 6h, and 9h. Moreover, we used wild-type B. subtilis directly reacting for 9h as Control group1 and transformed B. subtilis without naringrnin directly reacting for 9h as Control group2. For every single group, we set two different conditions---fracturing fluid and cracked liquid supernatant. Apart from the cofactors enzymes need to perform their functions, we also added Tris-HCl to control pH condition the same. And simultaneously, we simply repeated our first attempt to see whether it is operation caused errors.

We screened phages targeting the B. subtilis BS168 strain from the environment to achieve regulation of engineered bacteria. Further more, we hope to achieve dual regulation of the engineered bacteria, so we designed a component whose structure can be described as “lsl”, which stands for “loxp-terminator-loxp”. The two loxp component is in the same direction and the enzyme expressed by cre gene can combine to this component and cut off the stop,which is a terminator, so that perform the function of starting the gene expression process.

Objective: To sceen the phage targeting E. coli BL21 and B. subtilis BS168 strains from the environment.

Practice: We collected soil and water samples from the flower beds and lakes on the campus of Huazhong University of Science and Technology. These samples were co-incubated with the bacterial suspensions of E. coli BL21 and B. subtilis BS168 strains respectively for amplification. The phages were sceened using the Double Agar Overlay Assay and purified by the phage streaking method.

Conclusion: We successfully sceened phages targeting the E. coli BL21 strain and the B. subtilis BS168 strain from the soil, and obtained clear plaques.

Objective: To confirm the types of the phages we have selected and verify their antibacterial efficacy.

Practice: The concentration of phages was quantified by the Spot titer assay. The types of phages and their bactericidal effects were determined through Planktonic Bacteria Bactericidal experiment and Transmission Electron Microscopy(TEM).

Conclusion:

(1) The TEM electron-microscopy results indicated that the phage targeting the E. coli BL21 strain was mainly filamentous phage, namely lysogenic phage; while the phage targeting the B. subtilis BS168 strain was a combination of lysogenic and virulent phages.

(2) The Spot titer assay results showed that the Phage concentration (number of plaque forming units per milliliter, PFU/mL) of phage concentrated solution reached 10^9 PFU/mL.

(3) The Planktonic Bacteria Bactericidal experiment results proved that the phage had a good sterilization effect and could be used as a chassis organism for further modification.

Objective: To design a molecular switch that can make the whole production pathway controllable.

Practice: We used the method of chemical transformation to transform pETDuet-CHS-lsl-CHI+pCDF-cre into BL21 in chemically competent state. And we used BL21 transformed with pETDuet-CHS-lsl-CHI+pCDF as the Control group.

Objective: To see whether there is targeted protein to verify whether we obtained an effective cre-loxp structure.

Practice: We got the protein samples from engineering bacteria with different plasmids. The one with pCDF-cre is the Experiment group, while the one with pCDF is the Control group. Then we conducted SDS-PAGE measurement to see if we got our targeted protein.

Conclusion: The results of the SDS-PAGE measurement showed that both the Experiment and the Control group have targeted protein, which means that the terminator was cut off so that the targeted gene can be expressed. However, there should not be any targeted protein in the Control group. Two reasons can cause this phenomenon. Firstly, the terminator did not work out well.Secondly, the expression capacity of the target fragment is excessively strong, causing the repressor protein bound to the operon to lose its function. As a result, expression can occur without lactose induction.Therefore, in the future, we redesigned a plasmid by adding a rrNB terminator to solve the problem.

Objective: To verify whether the cre-loxp mechanism functions.

Experimental Procedure: Take the protein gel samples obtained after SDS-PAGE: engineered bacteria containing the pCDF-cre plasmid serve as the experimental group, and engineered bacteria containing the pCDF plasmid serve as the control group. Perform membrane transfer on the protein samples, add primary antibody and secondary antibody after blocking, then capture images for observation.

Conclusion:

(1) The TEM electron-microscopy results indicated that the phage targeting the Escherichia coli BL21 strain was mainly filamentous phage, namely lysogenic bacteriophage; while the phage targeting the B. subtilis BS168 strain was a combination of lysogenic and virulent phages.

(2) The Spot titer assay results showed that the Phage concentration (number of plaque forming units per milliliter, PFU/mL) of phage concentrated solution reached 10^9 PFU/mL.

(3) The Planktonic Bacteria Bactericidal experiment results proved that the phage had a good sterilization effect and could be used as a chassis organism for further modification.

We designed a plasmid to express Cry5Ba and Cry6Aa proteins in the C. elegans intestine. First, since the Cry proteins we used originated from Bacillus thuringiensis, heterologous expression in B. subtilis and E. coli might pose challenges. Therefore, we initially completed codon optimization. Subsequently, to enable high-level expression and purification for toxicity validation, we selected the pET28a vector from the pET series. The detailed design of our plasmid is outlined

Using homologous recombination technology, we designed homologous arms on the primers to recombine the gene with the vector.

The vector was linearized and subjected to PCR to obtain insertion fragments with homologous arms. Following gel verification, we performed gel recovery, followed by homologous recombination and transformation of competent DH5α cells. The resulting colonies were spread plate, picked individual bacterial colony, and individual bacterial colony PCR. PCR validation confirmed the presence of the desired band, which was subsequently sent for sequencing.

During preliminary background research, we discovered that Cry5Ba and Cry6Aa proteins exhibit non-overlapping mechanisms of action and demonstrate synergistic effects when treating wild-type nematodes. Therefore, we plan to design a fusion protein combining both to achieve enhanced insecticidal efficacy. To preserve their critical domains without disrupting the secondary structure of protein folding, we employed the flexible peptide GGGGS to link the two proteins. In our project, we designed two fusion proteins: one linked by (GGGGS)1 and another by (GGGGS)2.

Using overlap PCR technology, we designed primers with complementary overlapping sequences to link the two genes. Simultaneously, through homologous recombination, the fusion gene was recombined with the vector. Below are the primers we designed with complementary overlapping sequences.