Team:UGM Indonesia/Engineering

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Engineering

Engineering

Engineering

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Our main goal is to create a feasible on-off system of the cyanide production and degradation according to either the presence or absence of arabinose. This page displays three engineering cycles; i.e., cyanide production, cyanide degradation, and arabinose conversion. Afterwards, an engineering cycle about the Auviola on-off system is performed to associate the aforementioned three cycles. An engineering cycle about the bioreactor is also demonstrated as the implementation of our Auviola system.

HCN-regulating System

What are the properties of HCN production in C. violaceum?

The HCN synthase, or also known as glycine dehydrogenase, is an enzyme that facilitates the conversion of an amino acid glycine into cyanide.1 This enzyme is a heterotrimeric protein as encoded by a cluster of three genes called hcnABC operon. This enzyme belongs to the oxidoreductase class as it oxidizes the amine (CH-NH2) functional group into imine (C=NH) and consecutively cleaves the molecule into HCN and carbon dioxide (CO2).2

What are some concerns to be considered?

The resulted cyanide species were designed to dissolve the gold into the soluble coordination compound of (NaAu(CN)2) through the reaction below:

4Au + 8NaCN + O2 + 2H2O 4(NaAu(CN)2) + 4NaOH1

According to previous study, the gold leaching process requires HCN compounds with concentration of 600 mg/L.2 On the other hand, a wild-type of C. violaceum was previously studied to produce 0.71 - 16 micromoles/mL which nearly equals to 19 - 432 of HCN in 8 hours.3 Therefore, an optimization of produced HCN concentration should be conducted to achieve an effective gold leaching process.

What are the types of regulators tested in C. violaceum?

Two of several regulator genes have tested in C. violaceum are araC and tetR.4 The araC works dependably to arabinose level which acts as both an activator in the presence of arabinose and a repressor in the absence of arabinose.5 A PBAD is one of the inducible promoters presented in C. violaceum and regulated by the araC.6 On the other hand, tetR acts as a repressor and normally play a role in tetracycline resistance with PTET promoter.7

,8
Wetlab work: HCN Production

Our strategies for improving cyanide metabolism in the cells are to increase the accumulation of HCN, hydrolysis of HCN, and L-arabinose converter. In this study, the proposed design for increasing HCN production has been achieved. The principle behind this system is overexpression of hcnABC gene encoding cyanide synthase, an enzyme that is responsible for HCN synthesis, by addition of L-arabinose-induced pBAD-AraC regulon. The genetic circuit for this study is written in the Design page. At first, we were focusing on the first plasmid construction for the HCN production. To achieve this goal, several standard Biobrick parts as well as the hcnABC operon was required. Some standard parts that we use are listed below:

  1. BBa_K808000 (AraC-pBAD)
  2. BBa_B0015 (double terminator)
  3. BBa_B0034m (strong RBS)

The hcnABC operon as the main insert, were obtained through gene synthesis from Twist Bioscience. Subsequently, the plasmid map was created to determine the overall gene construction into the pSB1C3 vector. Benchling application system was used to design the circuit, particularly the HCN production plasmid (Kindly visit our Results page for our plasmid construct!).

We used AraC-pBAD derived from BBa_K1602055 to regulate the hcnABC operon. We modified the hcnABC operon by adding RBS before each gene (hcnA, hcnB, hcnC) and also double terminator B0015. The hcnB and hcnC genes have a high percentage of GC content so we faced some problems to synthesize it. Thus, we altered the hcnB gene sequence to reduce the GC content and to remove some illegal sites. We also altered some bases in hcnC gene to remove the illegal site. For a detailed description of the construction, please visit our Design page. The recombinant plasmid transformed was verified through colony PCR and the inserted parts were confirmed by sequencing analysis.

A Kinetic modelling for demonstrating an overexpression of cyanide was performed in Tellurium. This modelling used several parameters obtained from literature. In addition, genome-scale modelling was performed to optimize the metabolic flux of C. violaceum to achieve optimal cyanide.

Drylab work: Kinetic Modelling

Beside the wet lab, we also perform dry lab analysis to model the HCN production, HCN degradation, and L-Arabinose isomerase in engineered C. violaceum. We use kinetic modelling approach to perform this analysis.

Kinetic modelling of metabolic pathways is important for predicting the output of a metabolic process, and to understand what type of modification should be carried out if the output is not desirable. For our model, the complete equations are explained in the Model page. We collected most of our parameters from research papers and past iGEM team modelling.

Wetlab work: HCN Production

After we went through the design phase, we started to conduct our project at the Biotechnology Laboratory of Inter-University Center in Universitas Gadjah Mada. We ordered hcnA, hcnB, and hcnC sequences from Twist Bioscience. The sequences were referred to the genome of Chromobacterium violaceum ATCC 12472 (NCBI: GenBank: AE016825.1).

To meet the BioBrick standard, we altered some of the sequences i.e., hcnB and hcnC, to reduce the GC content and remove the illegal sites. However, Twist Bioscience reported a problem in synthesizing the hcnC gene, thus the hcnC synthesize failed. Thus, we isolated the hcnC gene using PCR from the genome of C. violaceum ATCC 12472 and removed an illegal site inside the sequence using overlap PCR (Phusion polymerase) (see Results page table 1, no. 7,8, and 9). The assembly of plasmid backbone (pSB1C3), AraC-pBAD (BBa_ K1602055), RBS (BBa_B0034m), hcnABC operon, and double terminator (BBa_B0015) were done using Gibson Assembly. The assembled constructs were then transformed into two strains of E. coli, BL21(DE3) and DH5α using standard heat shock protocol. The screening of transformats were selected on LB agar supplemented with Chloramphenicol for both strains.

Drylab work: Kinetic Modelling

The equations needed for modelling gold cyanidation are explained in our Model page. We were utilizing python in Jupyter with the Tellurium package for most of our model, except for Au and CN interaction.

Some of the kinetic constant cannot be found on any research paper or open source database that is available on the internet. Thus, we used the value of similar parameter(s).

Wetlab work: HCN Production

The resulting gene constructs were transformed into two chassisses and verified by colony PCR using VF2 VR primers. After the colony PCR and electrophoresis, the expected bands were incorrect in size and unobtainable. Our amplified insert using VF2 VR primers should be around 4600 bp, yet we merely obtain approximately 3000 bp band. On the upper side of some bands, ColA. There were thin bands of which the sizes are over 3000 bp, which indicated that the desirable construct was inserted to the chassis. Therefore, we subcultured the ColA colony to LB broth media supplemented with Chloramphenicol overnight for plasmid isolation (check the Results page for plasmid isolation result). For further confirmation, we send our isolated plasmid for sequencing. Now we are still waiting for the result of sequencing and we will update and report the result by the time we receive it.

Drylab work: Kinetic Modelling

The graph below shows concentration (M) vs time (second).

engineering-hcn-regulating-1
Wetlab work: HCN Production

It was such a challenge for us to construct the whole operon with the size over 4000 bp with high GC content. A more robust PCR method such as an adjustable KOD with high-fidelity PCR master mix kit is necessary to increase the PCR product and avoid point mutation that might happen in the amplified product.

Drylab work: Kinetic Modelling

We can learn that rhodanese was produced instantly, then the concentration of HCN decreased rapidly. The maximum speed of gold cyanidation only lasts briefly, less than 5 minutes. Biohydrometallurgy method is favored in mineral processing field due to their ability to extract low-concentrated ore. With this kind of trend, it is almost impossible to gain a feasible gold extraction yield. In lab experiments, most bioleaching processes managed to attain large mineral yield in laboratory experiments. This modelling might be not reflecting the real conditions of the bioleaching process.

Drylab work: Kinetic Modelling

We need to reconsider some of the parameters, especially those based on assumption. Rate of large organic molecule degradation is one of the main examples. There are 2 large organic molecules that are involved in this modelling, protein (enzyme) and monosaccharide. We need to separate the decay rate of protein and monosaccharide. SCN formation constant also needs to be changed. Due to larger scale use and relatively dynamic environment, we assumed that the collision rate would be significantly slower. Therefore, the constant also becomes lower. Rhodanese activates almost simultaneously after HCN hits its highest concentration, hence we need to set a starting point to delay rhodanese synthesis. The starting point will be based on L-arabinose concentration.

Drylab work: Kinetic Modelling

The parameter that we consider to be revised are:

Drylab work: Kinetic Modelling

The results of modelling using those parameters are:

engineering-hcn-regulating-2
Drylab work: Kinetic Modelling

The HCN concentration reduction becomes less steep, thus gold cyanidation could be more efficient. It will prolong the almost optimum gold leaching period and surely increase the potential gold yield. Although we achieved desirable results through modelling, further laboratory experiments also needed to verify our model and obtain more realistic results. Large scale and pilot testing are also required for industrial application.

HCN Degradation

These schemes below are our journey to degrade cyanide as the remaining compounds in the gold cyanidation based on the engineering approach to generate BBa_K4076002

What are the properties of HCN degradation in C. violaceum?

Interestingly, C. violaceum possesses several cyanide-utilizing enzymes–β-cyanoalanine synthase, -cyano-α-aminobutyric acid synthase, and rhodanese.9 These enzymes are briefly explained below:

  1. β-cyanoalanine synthase (β-CAS)

    This enzyme has been reported to facilitate the incorporation of cyanide into cysteine to form the less toxic amino acids or ammonia.10

  2. β-cyano-α-aminobutyric acid synthase

    This enzyme can catalyze the reaction between homocystine and cyanide to give the less toxic y- γ-cyano-α-aminobutyric acid and thiocyanate.11

  3. Rhodanese

    This enzyme is also known as sulfurtransferase, as it catalyzes sulfur transfer from thiosulfate to cyanide and leads to the formation of the less toxic thiocyanate.9,11

Among several cyanide-degrading genes, the rhodanese is chosen to be the added gene whose regulation is not affected by the presence of glycine and methionine, unlike the others.12

What are some concerns to be considered?

The higher HCN concentration may need a higher expression of its degradation enzyme. Therefore, an optimization of rhodanese expression should be conducted to effectively degrade HCN into its less toxic compound.

How to create a feasible HCN degradation part?

The principle in this system is induction of cyanide degradation in the absence of arabinose. In order to develop an on/off system between HCN production and degradation, we adopted pBAD-TetR regulation and use pTet to regulate the sseA gene to be expressed as sulfurtransferase or rhodanese enzyme. At this stage, we utilized several standard parts already used by previous iGEM teams listed below:

  1. BBa_C0080 (AraC)
  2. BBa_K542003 (pBAD-TetR)
  3. BBa_R0040 (pTet)
  4. BBa_B0034m (strong RBS)
  5. BBa_B0015 (double terminator)

We used pBAD-TetR (BBa_K542003) to regulate the sseA gene. The sseA gene has a high percentage of GC content so we faced some problems to synthesize it. Thus, we altered the sseA sequence to reduce the GC content and to remove some illegal sites. For a deeper understanding of the construction, you can visit the Auviola design page (linked to the design page).

We ordered the sseA sequence from Twist Bioscience. The sseA sequence referred to the genome of Chromobacterium violaceum ATCC 12472 (NCBI: GenBank: AE016825.1). In order to meet the BioBrick standard and synthesize requirement, we altered some of its sequences. The assembly of plasmid backbone (pSB1C3), AraC (BBa_C0080), pBAD-TetR (BBa_K542003), RBS (BBa_B0034m), sseA gene, and double terminator (BBa_B0015) were done using Gibson Assembly.

We also build a kinetic model using tellurium for cyanide degradation performed by rhodanese as a separate section model with parameters mentioned in previous studies.

On-going.

On-going.

L-Arabinose Converter

These schemes below are our journey to degrade cyanide as the remaining compounds in the gold cyanidation based on the engineering approach to generate --

What are the properties of regulator genes in C. violaceum?

Regulator genes play a crucial role in controlling an on-off system. Two of several regulator genes have been tested in C. violaceum are araC and tetR.4 The araC works dependably to arabinose level which acts as both an activator in the presence of arabinose and a repressor in the absence of arabinose.5 A PBAD is one of the inducible promoters presented in C. violaceum and regulated by the araC.6 On the other hand, tetR acts as a repressor and normally play a role in tetracycline resistance with PTET promoter.7,8 These regulator genes as well as their related promoters were the candidates for controlling our Auviola system.

What are some concerns to be considered in C. violaceum engineering?

Since C. violaceum is unable to ferment arabinose, a system responsible for converting arabinose into its inactive form is needed. An enzyme of L-arabinose isomerase belongs to the L-arabinose converter into L-ribulose, so that the arabinose concentration can be adjusted.13

On-going.

On-going.

On-going.

On-going.

Pyrite Dissolution Bioreactor

References

  1. KEGG, ENZYME: 1.4.99.5 [Online] https://www.genome.jp/dbget-bin/www_bget?ec:1.4.99.5 [accessed on July 28, 2021 at 09:06 WIT].
  2. Kianinia, Y., Khalesi, M.R., Abdollahy, M., Hefter, G., Senanayake, G., Hnedkovsky L., Darban, A.K., Shahbazi, M., 2018, Predicting Cyanide Consumption in Gold Leaching: A Kinetic and Thermodynamic Modeling Approach, Minerals, vol. 8, no. 3, pp. 110. doi:10.3390/min8030110
  3. Michaels, R. and Corpe, W.A., 1965, Cyanide Formation by Chromobacterium violaceum, Journal of Bacteriology, vol. 89, no. 1, pp. 106-112.
  4. Brazilian National Genome Project Consortium, 2003, The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability, Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 20, pp. 11660-11665.
  5. Lobell, R.B., Schleif, R.F., 1990, DNA looping and unlooping by AraC protein, Science, vol. 250, no. 4980, pp. 528-532.
  6. Schleif, R., 2003, AraC protein: a love-hate relationship, Bioessays, vol. 25, pp. 274-282.
  7. Brazilian National Genome Project Consortium, 2003, The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability, Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 20, pp. 11660–11665. doi:10.1073/pnas.1832124100
  8. Cuthbertson, L., Nodwell, J.R., 2013, The TetR family of regulators, Microbiology and molecular biology reviews: MMBR, vol. 77, no. 3, pp. 440–475. doi:10.1128/MMBR.00018-13
  9. Machingura, M., Salomon, E., Jez, J.M., Ebbs, S.D., 2016, The β-cyanoalanine synthase pathway: beyond cyanide detoxification, Plant, Cell and Environment, vol. 39, no. 10, pp. 2329-41.
  10. Ressler, C., Abe, O., Kondo, Y., Cottrell, B., Abe, K., 1973, Purification and Characterization from Chromobacterium violaceurn of an Enzyme Catalyzing the Synthesis of -Cyano-a-aminobutyric Acid and Thiocyanate, Biochemistry, vol. 12, no. 26, pp. 5369-5377.
  11. Cipollone, R., Ascenzi, P., Tomao, P., Imperi, F., Visca, P., 2008, Enzymatic Detoxification of Cyanide: Clues from Pseudomonas aeruginosa Rhodanese, Journal of Molecular Microbiology and Biotechnology, vol 15, pp. 199-211.
  12. Rodgers, P.B., Knowles, C.J., 1978, Cyanide Production and Degradation During Growth of Chromobacterium violaceum, Journal of General Microbiology, vol. 108, pp. 261-267.
  13. Uniprot, 2021, UniProtKB - P08202 (ARAA_ECOLI) [Online] https://www.uniprot.org/uniprot/P08202 [accessed on August 17, 2021 at 12:25 WIT].
  14. Jones, C.A., Kelly, D. P., 1983, Growth of Thiobacillus ferrooxidans on ferrous iron in chemostat culture: Influence of product and substrate inhibition, Journal of Chemical Technology and Biotechnology: Biotechnology, vol. 33, no, 4, pp. 241-261. doi:10.1002/jctb.280330407
  15. Suzuki, I., Lizama, H.M., Tackaberry, P.D., 1989, Competitive Inhibition of Ferrous Iron Oxidation by Thiobacillus ferrooxidans by Increasing Concentrations of Cells, Applied and Environmental Microbiology, vol 55 no 5, pp. 1117–1121. doi:10.1128/aem.55.5.1117-1121.1989