We tried to create an on-off system for HCN production and HCN degradation for C. violaceum. We engineered 3 plasmids which consist of the hcnABC operon (the HCN production plasmid combined with AraC-pBAD promoter), the rhodanese gene (HCN degradation gene with AraC repressible promoter), and L-arabinose isomerase (responsible to transform the Arabinose to shut down the HCN production). The overview of our gene construct depicted in Figure 1. Due to the complexity of the project and the construct itself, we divided our project into 3 sub-projects and for this time we are focusing to create the first construct, which is the HCN production circuit.
Gene construction of for HCN production
To construct the HCN production gene, we needed several biobrick parts. Gene construction for HCN production consists of several parts:
- AraC-pBAD promoter (BBa_K1602055)
- RBS (BBa_B0034)
- hcnABC operon (synthesized by Twist Bioscience)
- hcnC from extracted genome C. violaceum
- Double terminator (BBa_B0015)
- BBa_B0034 (strong RBS)
- Origin of replication from pBBR1-MCS2 (obtained from colleague)
The total legth of the overal construct with the backbone is around 7000s bp (Figure 2). During the first step of the assembly, we used the native origin of replication from pSB1C3.
Each of the required part was amplified by Phusion High-Fidelity PCR Kit with the specific primers as shown in Table 1. The gene amplification was visualized by gel electrophoresis under UV-light (Figure 3) and purified by gel purification system in order to obtain the desirable gene sequence. The DNA concentration of each product were quantified using Nanodrop (Table 2).
Gel purification was performed for sample 1, 2, 5, 6, 8, 9, 10, and 11. The rest samples were purified using PCR clean-up purification protocol.
|Tube||DNA Concentration (ng/uL)|
After all of the constructs with the overhang sites (elongated through PCR system) were obtained, then we continued the process for the assembly. Some constructs that we assembled consist of:
- P1: BBa_ K1602055 (Biobrick Parts)
- P2: hcnAB (Twist Bioscience)
- P3: hcnC-1st fragment (isolated from C. violaceum WT genome; before the illegal site)
- P4: hcnC-2nd fragment (isolated from C. violaceum WT genome; after the illegal site)
- P5: BBa_B0015 double terminator, together with the backbone
These parts were all assembled using the Gibson assembly method. The reaction mix are incubated in 50oC for 1 hour in a thermal cycler.
We transformed the construct to E. coli \(BL21(DE3)\) and \(DH5α\) using a strandard heat shock transformation protocol. LB agar plate supplemented with chloramphenicole was used to select the transformants. After an overnight incubation, we did the colony PCR, using VF2 VR primers (Table 3), to the randomly picked the colonies and directly subcultured the colonies and subcultured them in the LB broth with antibiotic. The following figure (Figure 3) depicts the results.
The elctropherogram showed that the DNA size were merely under 1500 bp, however, there were some bands appears with the size of arround 4600 bp. We suspected that the PCR mix was not robust to amplify our construct. Therefore, we directly subcultured the first colony (ColA) for plasmid extraction, obtaining the concentration of ColA: 146.20 ng/uL.
Another PCR cycle was conducted using hcnABC primers to confirm whether the construct is fully inserted to the vector (Figure 4).
Additional bands were shown on the gel electrophoresis result with the size of around 5000 bp and 1500 bp. Although the desired band (~4600s bp) have been obtained, further investigation is necessary in order to confirm that the construct is correct. Therefore, we send the extracted plasmid and some primers for Sanger sequencing analysis.
This time we still waiting for the sequenced plasmid. The sequencing result will be reported soon.
By the time we receive the sequencing result, we will directly assess the construct and measure the accumulation of cyanide production in E. coli \(BL21(DE3)\) expression system. In contrast, the assembly and reconstruction will be conducted when the result were not as we expected. Besides, we also plan to change the ORI from pBBR1-MCS2 plasmid because this ORI is compatible for both E. coli and C. violaceum.1 Next, we will transform the plasmid to competent cell C. violaceum using some methods such as heat shock, optimized electroporation,2and freeze thawing method. Transformation optimization is necessary for this step because C. violaceum \(ATCC 12472\) is a non-model chassis, which makes it more challenging engineer.
Following that, we plan to measure the cyaide production of the engineered C. violaceum \(ATCC 14272\) using picric acid method,3 confirm and re-assess our Genome Scale Model and kinetic models, and compare the cyanide production with the Wild Type. If the result found out that the average cyanide production is higher, it will be the first step for further improvement, i.e., pH optimization, given that the bioleaching process by cyanide is a pH-dependent process.
- Liow, L.T., Go, M.K., Chang, M.W., Yew, W.S., 2020, Toolkit development for cyanogenic and gold biorecovery chassis Chromobacterium violaceum, ACS Synth. Biol, vol. 9, pp. 953 – 961.
- Loke, W.K. and Saud, H.M., 2020. Electrotransformation on selected plant growth-promoting Rhizobacteria (PGPR) and Chromobacterium violaceum. Journal of Agrotechnology, vol. 11, no. 1, pp. 63 – 69.
- Drochiou, G., Pui, A., Danac, R., Basu, C., Murariu, M., 2003, Improved spectrophotometric assay of cyanide with picric acid, Revue Roumaine de Chimie, vol. 48, no. 8, pp. 601 – 606.