Engineering Success

Design

The concept of our project, to insert genes that are known to degrade Polychlorinated biphenyls into an organism, was inspired by Stockholm's 2020 project. In their project, they inserted their genes into yeast, but we were unsure about yeast’s capabilities as a chassis for use in waterways. We researched other organisms to possibly use by reading literature before settling on the Rhodococcus bacteria. Rhodococcus, while already displaying a natural inclination to degrade PCBs, can also survive in aerobic and anaerobic environments, which would be useful for treating PCB contamination in all environments. It has also been shown in literature that foreign genes can be inserted into Rhodococcus.

A difficulty we identified in the engineering process was that the term PCBs describes a wide variety of chemical congeners with differing amounts and positions of chlorines on phenyl rings. Commercial mixtures of PCBs, known in the United States as Aroclors, are complex mixtures of 60–90 types, congeners, of PCB molecules that differ in the number and position of chlorines on the phenyl rings. However, we found in literature that the genes PCBA1, PCBA4, and PCBA5 of the dehalogenese strains have been found to naturally degrade many different congeners of PCBs [1]. Each of the genes produce enzymes that target different chlorines in different positions and each targets dozens of the congeners making them an effective dechlorination force. In response to these findings, we decided to insert these three genes into our chassis of Rhodococcus.

We decided to insert these genes into Rhodococcus using pSB1C3 as a vector. We thought this vector, being a standard vector for iGEM, would be a good place to start when engineering since we could not find adequate sources for other Rhodococcus vectors.

Build

To build our plasmids we performed these steps:

1. Identify the genes in the article that explained the genomic characterization of the three unique Dehalococcoides [2]
2. Looked for the specific portion of the genome in Genbank
3. Exported the gene to Benchling
4. Performed codon optimization for Rhodococcus using the Genscript tool [3]
5. Made sure to remove restriction sites: Xbal, Pstl, Spel, EcoR1
6. Found a promoter used in the paper, An Integrative Toolbox for Synthetic Biology in Rhodococcus, PM6-OP3 [4]
7. Used a common RBS, BOO34
8. Used a common terminator that was also used by the 2016 Edinburgh team that worked on PCBs, B0015

Test

Once we received the genes from IDT, we performed ligations and transformations with them. However, no successful colonies were created. We resuspended, ligated, and transformed the genes again. Once more, no successful colonies were created.

Iteration

We therefore redesigned our system so that instead of using pSB1C3, we used the SmartJoin vector which accepts any blunt DNA piece. Now when we resuspended, ligated, and transformed the genes into E. coli we got colonies. We screened them by colony PCR and got a band of the correct size, which shows that our engineering worked. We are preparing to transform the plasmids into Rhodococcus now to show that they work there also.

Design

Present methods of PCB detection require long lengths of time; the real-time monitoring of sites must be accessible and efficient to minimize exposure throughout the food chain. Also, large efforts for the detection of PCBs occur only in largely contaminated sites in the status quo, leaving PCBs in areas with undetected quantities to pile up [5]. PCBs are difficult and expensive to measure because the composition of the PCBs changes over time, meaning that the relative level or amount of PCBs present in a certain space also changes over time. Since the composition changes, the toxicity can also change, making it hard to get a consistent and accurate reading. [6]

PCBs are most commonly detected using gas chromatography-mass spectrometry. This technique is considered the gold standard because it can explicitly identify organic molecules in complex mixtures. Gas chromatography-mass spectrometry needs a high vacuum, which requires maintenance, which is time consuming and expensive. It also requires organic solvent extraction to concentrate PCBs for its analysis, which can have additional environmental and health risks [7]. Limitations of these methods are that they are based on the availability of materials and facilities with gas chromatography columns or complex chemical analytical techniques. They determine the total concentration of pollutants, which overestimates the risk of impact on living organisms as only a fraction of the total amount of pollutant is the bioavailable fraction; the inability to distinguish between the two fractions of pollutants poses a major disadvantage to current, expensive, and time-consuming techniques.

The recent practice of biosensing by utilizing synthetic biology and the advantages of fusing reporter genes, such as lux, gfp, or lacZ, to responsive promoters and has begun to be used for PCB detection. In 1998, Layton et al. reported a bioluminescent reporter strain, Ralsonia eutropha ENV307 (pUTK6), detecting the bioavailability of PCBs through inserting the biphenyl promoter directly upstream of the bioluminescence genes [8]. All such biosensors are used in vitro and in environments such as soil.

We searched for a new, efficient, and accurate method of biosensing through taking advantage of the dioxin-responsive elements (DRE) of the Murine CYP1A1 gene promoter and the expression of the human aryl hydrocarbon receptor complex in yeast (AHR). Ultimately, luciferase will be produced and measured to detect PCBs quickly and efficiently.

Sensing
To detect PCBs, we created a sensor containing:

• Bidirectional promoter between yeast GAL1 and GAL10 genes
• Promoter regulates expression of complex: AhR (Human Aryl Hydrocarbon Receptor) and ARNT (Aryl Hydrocarbon Receptor Nuclear Translocator)
• The complex translocates to nucleus when it binds PCBs

The human AHR complex and hydrocarbon receptor nuclear translocator protein (ARNT) have been coexpressed in the yeast, Saccharomyces cerevisiae, with specific transcription activation assessed by β-galactosidase activity produced from a reporter plasmid [9]. Coexpression of human AHR and ARNT lead to the transcription of a DRE-containing reporter plasmid in the absence of exogenous AHR agonists. Critically, the majority of PCB congeners are likely to interact with AHR in their non-planar conformations, meaning that they are dioxin-like and are likely to be recognized as dioxins by the AHR complex. When activated by a dioxin, or, in the case of the study, a dioxin-like PCB, the AHR translocates into the nucleus and dimerizes with the ARNT protein to form the AHR/ARNT complex, which then binds to a specific DNA recognition site within a nucleus: the DRE.

Response
To respond to the signal:

• AhR/ARNT complex in the nucleus binds to 7 DREs (DNA recognition site) in the CYP1A1 promoter
• CYP1A1 promoter activates CYP1A1 gene fused to the Akaluc luciferase reporter

When activated by dioxin, the cytosolic AhR protein complex translocates into the nucleus and dimerizes with the ARNT (Ah receptor nuclear translocator) protein. The heteromeric ligand:AhR/Arnt complex then recognizes and binds to its specific DNA recognition site, the dioxin response element (DRE). DREs are located upstream of cytochrome P4501A1 (CYP1A1) and other AhR-responsive genes, and binding of the AhR complex stimulates their transcription. Although CYP1A1 expression has been used as the model system to define the biochemical and molecular mechanism of AhR action, there is still limited knowledge about the roles of each of the seven DREs located in the CYP1A1 promoter.

Build

To construct our device, we used Gibson Assembly to create our final plasmid pLK001 that contained:

• Digested pBF3038
• CYP1A1 gene fragments
• Human CYP1A1 promoter region
• Akaluc

Test

We assembled our parts using Gibson Assembly and were able to successfully get colonies!







Iteration

[1] Bedard, D. L. (2014). PCB dechlorinases revealed at last. Proceedings of the National Academy of Sciences, 111(33), 11919–11920. https://doi.org/10.1073/pnas.1412286111

[2] Wang, S., Chng, K. R., Wilm, A., Zhao, S., Yang, K.-L., Nagarajan, N., & He, J. (2014). Genomic characterization of three unique dehalococcoides that respire on persistent polychlorinated biphenyls. Proceedings of the National Academy of Sciences, 111(33), 12103–12108. https://doi.org/10.1073/pnas.1404845111

[3] GenSmart™ Codon Optimization Tool-GenScript. (n.d.). Retrieved October 14, 2022, from https://www.genscript.com/gensmart-free-gene-codon-optimization.html

[4] Round, J. W., Robeck, L. D., & Eltis, L. D. (2021). An integrative toolbox for synthetic biology in Rhodococcus. ACS Synthetic Biology, 10(9), 2383–2395. https://doi.org/10.1021/acssynbio.1c00292

[5] Chobtang, J., De Boer, I. J., Hoogenboom, R. L., Haasnoot, W., Kijlstra, A., & Meerburg, B. G. (2011). The need and potential of biosensors to detect dioxins and dioxin-like polychlorinated biphenyls along the milk, eggs and meat food chain. Sensors, 11(12), 11692–11716. https://doi.org/10.3390/s111211692

[6] Hornbuckle, K., & Robertson, L. (2010). Polychlorinated biphenyls (PCBS): Sources, exposures, toxicities. Environmental Science &Technology, 44(8), 2749–2751. https://doi.org/10.1021/es100801f

[7] Liu, X., Germaine, K., Ryan, D., & Dowling, D. (2010). Whole-cell fluorescent biosensors for bioavailability and biodegradation of polychlorinated biphenyls. Sensors, 10(2), 1377–1398. https://doi.org/10.3390/s100201377

[8] Miller, C. A. (1997). Expression of the human aryl hydrocarbon receptor complex in yeast. Journal of Biological Chemistry, 272(52), 32824–32829. https://doi.org/10.1074/jbc.272.52.32824

[9] Environmental Protection Agency. (2019, October 4). Guidance for Applicants Requesting to Treat/Dispose of PCBs Using Incineration or an Alternative Method. Regulations.gov. Retrieved October 13, 2022, from https://www.regulations.gov/document/EPA-HQ-OLEM-2018-0305-0010