Engineering

1. Formaldehyde indicating system

Design

FrmR is a repressor protein from E. coli. In the absence of formaldehdye, FrmR binds to the promoter region, preventing transcription. In the presence of formaldehyde, the nucleophilic Cys36 of the FrmR reacts with formaldehyde and causes a conformational change that results in dissociation from the promoter^[1]. Based on this, we can construct the downstream gene expression system induced by formaldehyde through FrmR and corresponding promoter sequences（figure 1）.

In order to improve the sensitivity of E. coli to formaldehyde, we selected the promoter pTR47M4 optimized by Woolston, BM. et al^[1]. The downstream gene was then placed with sfGFP, a high-brightness green fluorescent protein. The genome company was commissioned to synthesize the entire sequence and construct it into the skeleton plasmid pET28a. Here is our plasmid map (figure 2):

Build

After receiving the synthetic plasmid from a genetics company, the plasmid is transformed into E. coli B21 (DE3). And we performed colony PCR validation（figure 3） for checking if transformation succeed. The PCR products were sent to a gene company for further sequencing verification.

Test

We added formaldehyde solution to E. coli strain 1aI cultured overnight, continued to culture for one hour, and observed under a fluorescence microscope, and found that E. coli emitted obvious green fluorescence.

We added formaldehyde solution with different concentration gradients into the bacterial solution of E. coli strain 1aI (OD≈0.4) at the beginning of the log stage, and read the fluorescence signal every 5 minutes in the microplate reader（figure 5）.

Learn

We found that under the induction of formaldehyde, the fluorescence intensity of E. coli strain 1aI increased with time and gradually reached saturation. At the same time, the fluorescence intensity increased with the increase of concentration in the range of 0-250μM. After the concentration is greater than or equal to 500μM, the cell no longer grows, the fluorescence no longer increases, and E. coli strain 1aI may not be able to tolerate formaldehyde above this concentration.

2. Formaldehyde metabolic system

Design

Formaldehyde is a very common secondary metabolite in living organisms, so many organisms have the ability to metabolize formaldehyde, which also includes our chassis microbes - E. coli. However, the formaldehyde metabolism capacity of wild-type E. coli is not enough to meet our needs. Therefore, we decided to transform formaldehyde dehydrogenase and formate dehydrogenase into E. coli BL21 (DE3) to improve the ability of E. coli to metabolize formaldehyde.

First cycle

Design

At first, for formaldehyde dehydrogenase, we chose fdh gene from Methylorubrum extorquens^[2].

fdh(formaldehyde dehydrogenase) protein can oxidize formaldehyde to formic acid in two steps. In the first step, reduced glutathione(GSH) reacts with formaldehyde in an enzyme-independent manner to form S-hydroxymethyl glutathione, which is then transported to the cell solute, S-Formylglutathione is formed as a substrate of fdh. Then S-Formylglutathione is hydrolyzed by hydrolase to form formic acid and glutathione.^[2]

For formate dehydrogenase, we referred to the 2019 iGEM team ZJUT-China and chose lbfdh(BBa_K2936004) gene from Lactobacillus buchneri they used.

We placed fdh gene and lbfdh gene after P_FrmR promoter and FrmR gene(figure 6), so that in the absence of formaldehyde, corresponding proteins would not be expressed to affect the growth of E. coli itself. In the presence of formaldehyde, formaldehyde dehydrogenase and formate dehydrogenase are expressed to completely oxidize formaldehyde to carbon dioxide and water to complete the removal of formaldehyde.

Here is our plasmid map(figure 7):

Build

After receiving the synthetic plasmid from a genetics company, the plasmid is transformed into E. coli B21 (DE3). And we performed colony PCR validation for checking if transformation succeed. The PCR products were sent to a gene company for further sequencing verification.

Test

We tested the formaldehyde metabolizing ability of E. coli strain 2b(with pET28a-FrmR-fdh-lbfdh ) and wild-type E. coli BL21(DE3). (figure 8)

Learn

As is shown in the figure, the results of this attempt are not satisfactory. The metabolic capacity of E. coli with the gene related to formaldehyde metabolism was lower than that of wild-type E. coli. By detecting the pH value of the medium, we found that after overnight culture, the pH of the medium of strain 2b decreased significantly (ph≈6). Therefore, we speculated that the reason for the slow metabolism was that the oxidation rate of formic acid was slower than that of formaldehyde, resulting in the accumulation of formic acid, which reduced the pH of the system and the activity of E. coli. Based on this, we made a second try.

Second try

Design

We chose CboFDH to replace lbfdh(figure 9).

CboFDH is a kind of NAD+-dependent formate dehydrogenase from Candida boidinii, which could catalyzes the reversible conversion of formate to carbon dioxide using as coenzyme the NAD+/NADH system^[3]. We selected CboFDH optimized after Site-saturation mutagenesis by Weihua Wu et al.^[4], which has a higher catalytic efficiency.

Here is our plasmid map（figure 10）:

Build

After receiving the synthetic plasmid from a genetics company, the plasmid is transformed into E. coli B21 (DE3). And we performed colony PCR validation for checking if transformation succeed. The PCR products were sent to a gene company for further sequencing verification.

Test

We tested the formaldehyde metabolizing ability of E. coli strain Cb(with pET28a-FrmR-fdh-CboFDH) and wild-type E. coli BL21(DE3).(figure 11)

Learn

As is shown in figure 11 , we achieved a satisfactory result this time. Cb has stronger formaldehyde metabolism capacity than wild-type E. coli BL21(DE3). Cb removed 93.0% of formaldehyde within 30min, while wild-type E. coli BL21(DE3) without the corresponding gene only removed 56.7% of formaldehyde. We succeeded in increasing the ability of E. coli to metabolize formaldehyde.

3. Hydrogen sulfide indicating system

Design

We selected 3 genes: sqrR, SQR, mCherry for this system.

SqrR is a repressor protein. In the absence of hydrogen sulfide, C41 and C107 of SqrR are in the reduced form. This form of SqrR binds the promoter region and represses the expression of downstream gene. In the presence of hydrogen sulfide, the resultant cellular RSS(highly oxidized sulfur species,termed reactive sulfur species) promotes the formation of a di-, tri-, or tetrasulfide bond between C41 and C107, inhibiting the ability of SqrR to bind to the promoter region. RNA polymerase binds DNA and subsequently induces gene expression^[5].

SQR(Sulfide quinone oxidoreductase) is a disulfide oxidoreductase, which oxidizes S^2- to zero-valent S(S_n). And S_n inhibits the ability of SqrR to bind to the promoter region as a kind of RSS, then downstream genes were expressed.

mCherry is a red fluorescent protein that is widely used in biotechnology as a tracer. In our scenario, in the absence of H₂S, the sqrR protein binds to the promoter region and prevents downstream mCherry expression; When hydrogen sulfide is present, SQR oxides S^2-, generates S_n, prevents sqrR from binding to the promoter, and the downstream mCherry is normally expressed and emits red fluorescence.

Here is our plasmid map（figure 12）:

Build

After receiving the synthetic plasmid from a genetics company, the plasmid is transformed into E. coli B21 (DE3). And we performed colony PCR validation（figure 12） for checking if transformation succeed. The PCR products were sent to a gene company for further sequencing verification.

Test

Unfortunately, the hydrogen sulfide indicating system we designed did not work as we expected. After we added sodium sulfide, the E. coli didn't turn red as expected.

Learn

After talking with the team members and our Instructor, we suspect that the reason may be that the expression of the SQR gene is too low to activate the downstream pathway.

To test this hypothesis, we cultured H2S indicated E. coli strain M with another E. coli strain SS(with SQR gene) overnight after adding sodium sulphide. After that, the bacterial solution was made into a temporary slide and observed under a fluorescence microscope, and it was successfully found that some E. coli cells emitted red fluorescence(figure 13). And it was often broken E. coli cell that glowed intensely red under the fluorescence microscope, which confirmed our conjecture.

4. Hydrogen sulfide metabolic system

Design

In this system, we refer to the work of Team Tongji_China in 2021 and make modifications on this basis. We selected two genes from Tongji_China(2021), SQR(BBa_K3823001) and SDO(BBa_K3823002).

As mentioned above, SQR can oxidize S^2- to Sn. Then SDO(Sulfur dioxygenase) is a GSH-dependent homodimer and the molecular mass of each subunit is approximately 23 kDa. It could catalyze the oxidation of S0 to sulfite in the presence of reduced glutathione (GSH) by using Fe³⁺ or molecular oxygen as electron acceptor.

Different from Tongji_China(2021), we selected two other enzymes to oxidize sulfite to sulfate, which are sorA and sorB.

SOR(sulfite:cytochrome c oxidoreductase) is the main enzyme system for oxidizing sulfites in bacteria. It is encoded by two genes, sorA and sorB, and generally forms heterodimeric. SorA is a large subunit containing Mo (about 40.2kDa), while SorB is a small subunit containing Heme c. It's only 8.8 kDa. SorAB is a kind of SDH(sulfite dehydrogenases) distributed in periplasmic space. SorAB in bacteria can oxidize sulfites directly to sulfate^[6]. We selected sorA (GenBank accession number AAZ62443.1) and sorB (GenBank accession number AAZ62442.1) genes from Cupriavidus pinatubonensis JMP134^[6].

Here is our plasmid map（figure 15）:

Build

After receiving the synthetic plasmid from a genetics company, the plasmid is transformed into E. coli B21 (DE3). And we performed colony PCR validation for checking if transformation succeed. The PCR products were sent to a gene company for further sequencing verification.

Test

Unfortunately, the hydrogen metabolic indicating system we designed did not work as we expected.

5. co-culture system

5.1 Sucrose production

Design

In order to keep the metabolic system running continuously, we added engineered Synechococcus elongatus to the system as producers.

When S. elongatus are cultured in an environment with high salt concentration, they can synthesize and secrete sucrose as an osmolyte. However, S. elongatus do not actively release sucrose outside the cell. On this basis, S. elongatus were designed to express sucrose permease(CscB), a sucrose/proton symporter, which could enable S. elongatus to secrete sucrose into the growth medium and support the growth of E. coli in the absence of other carbon sources^[7].

We collaborated with the iGEM team shanghaitech-china(2022), who generously donated the plasmids from their project last year. Here's a map of the plasmids they shared with us (Figure 16).

Build

After receiving the plasmids shanghaitech-china(2022) shared with us, the plasmid is transformed into S. elongatus PCC7942.

Test

Unfortunately, we only transformed the cscB gene into S. elongatus two days before the freeze day because S. elongatus PCC7942 were contaminated with other bacteria. So we didn't have time to test our strain.

Learn

The slow growth rate of S. elongatus PCC7942 is the main reason that restricts the progress of our experiment. We learned that another S. elongatus strain, UTEX2973, has a very similar genome and traits to PCC7942, but has a faster growth rate and is a better territorial microbe. However, unfortunately, there are no commercial companies selling this strain in China mainland. So we didn't get this strain.

5.2 Sucrose decomposition

Design

Because wild-type E. coli did not have good use of sucrose, we have expressed a β-fructofuranosidase (SacC, BBa_K4115017) in E. coli, after referring to the scheme of Team shanghaitech-china(2022). SacC is secreted into the intercellular space to hydrolyze sucrose into glucose and fructose. Then these two simple sugars can then be efficiently absorbed and utilized by E. coli.

Here is our plasmid map(figure 17):

Build

After receiving the synthetic plasmid from a genetics company, the plasmid is transformed into E. coli B21 (DE3). And we performed colony PCR validation for checking if transformation succeed. The PCR products were sent to a gene company for further sequencing verification.

Test

E. coli with SacC gene and wild-type Escherichia coli were cultured in LB medium containing the same concentration of sucrose, and the sucrose concentration in the medium was measured every 1.5h(figure 18).

Learn

The concentration of sucrose in culture medium of E. coli with SacC gene decreased obviously, indicating that E. coli was absorbing and utilizing sucrose. However, the sucrose concentration in the culture medium of wild-type E. coli increased, which was unreasonable, because E. coli itself did not produce sucrose. We speculated that E. coli produced some metabolites with similar structure to sucrose in the metabolic process, which affected the measurement of sucrose concentration by the kit.

Reference

[1] Benjamin M. Woolston, et. al. (2018) Development of a formaldehyde biosensor with application to synthetic methylotrophy. Biotechnology and Bioengineering. 115:206–215.

[2] 马凯 (2022) 甲醛高效降解菌的筛选驯化及其关键酶的研究(安徽农业大学)

[3] Bulut, H., et. al. (2021) Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase. Applied Biochemistry and Biotechnology. 193:363–376.

[4] Weihua Wu et al (2019) Site-saturation mutagenesis of formate dehydrogenase from Candida bodinii creating effective NADP+-dependent FDH enzymes. Journal of Molecular Catalysis B: Enzymatic. Volume: 61 157–161.

[5] Shimizu, T., et al (2017) Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis. PNAS. Volume:114 Issue:9 Page:2355-2360 DOI10.1073/pnas.1614133114.

[6] Yufeng Xin., et al(2020) The Heterotrophic Bacterium Cupriavidus pinatubonensis JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate. Applied and Environmental Microbiology. Volume:86 Issue:22 e01835-20.

[7] Cui, YX., et al (2022) Construction of an artificial consortium of Escherichia coli and cyanobacteria for clean indirect production of volatile platform hydrocarbons from CO₂. FRONTIERS IN MICROBIOLOGY volume: 13. DOI: 10.3389/fmicb.2022.965968.