Team:Chalmers-Gothenburg/Design

Design of Parts

One of the most important steps during the early stages of the project was designing our BioBricks. This meant identifying which genes to use and determining how genes, promoters and terminators should be combined to create the composite BioBricks. Additionally, the method by which the parts should be assembled had to be determined, to design the appropriate primers. Below is a description of this design process.

Selection of Genes

The team knew at a very early stage that we wanted to do a project that aimed to improve the environment in some way. While brainstorming project ideas, the issue of PCBs in the environment came up as a promising topic, around which a project could be based. Further research about the topic revealed several microorganisms that had shown the capacity to break down PCB to various extents, both bacteria and eukaryotes [1, 2].

The first thing that had to be decided was which enzymes to use to construct a PCB degradation pathway. While there initially was an interest in heterologously expressing genes from white-rot fungi that have shown the ability to degrade PCB, such as Pleurotus ostreatus [2, 3], there is a lot of uncertainty regarding the actual degradation mechanisms that these fungi use [4]. For this reason, it was decided to use enzymes from PCB degrading bacteria instead, since the functions of these are better characterized [5].

Eleven genes were identified from the biphenyl degradation pathway, with the best characterized homologs found coming from Rhodococcus jostii, Pseudomonas pseudoalcaligenes and Parabukholderia xenovorans [5]. This pathway has shown the capacity to metabolize biphenyl, along with certain lowly chlorinated PCB congeners, largely due to a dechlorinating enzyme being a part of the pathway in some organisms [5, 6]. However, in order to more efficiently degrade the PCBs that are present in the environment, which includes several types of highly chlorinated congeners [7], we wanted to add another enzyme to the pathway. Specifically, this was the reductive dehalogenase enzyme PcbA5, from the bacteria Dehalococcoides mcartyi, which has been shown to be able to dechlorinate a wide range of congeners [8], converting them into lowly chlorinated congeners that could then be metabolized by the biphenyl degradation pathway.

Figure 1. Illustration of the bacteria from which the genes used to create the PCB degradation pathway originate.

With the plan laid out, we realized that it would not be easy to heterologously express twelve genes in a new chassis organism. With this in mind, mathematical modeling was used to investigate the possibility of removing some of the genes, without losing too much efficiency during the degradation process. The details of this process can be found on the modeling page. After taking the results from the modeling into account, it was decided to condense the biphenyl degradation pathway down to eight genes, leaving out the last three enzymes in the pathway, which meant that the total number of genes that were to be integrated into the new host were nine.

Choice of Chassis Organism

After identifying the genes that would allow for PCB degradation, the next step was deciding what organism to use as a chassis. In order to make the project as simple as possible, the team wanted to use a well characterized organism, for which a lot of tools are available. For this reason, the choice was between the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. Eventually the team decided to use S. cerevisiae, for reasons including that this kind of project has never been attempted in yeast, to our knowledge, and that the laboratory where we would work specializes in working with yeast. After this was decided, the gene sequences that were to be ordered were codon optimized for S. cerevisiae. In addition, a variant of each gene, with a tag (either His-tag or FLAG-tag), was designed with the intent of using these to test whether S. cerevisiae is able to express these genes.

Dual Promoter Design

Due to the number of genes that we planned to integrate into the genome of S. cerevisiae being quite large, we devised a plan to integrate two genes at each integration site. This meant that after construction of the proper DNA fragments, only five genome integrations, using CRISPR/Cas9, would have to be performed. To this end, we designed a set of dual promoters by combining known strong yeast promoters, with the intent being that the two genes that were to be integrated at the same site in the genome would be transcribed in opposite directions along the chromosome, as shown in Figure 2. Four such dual promoters were designed, one for each integration site that would harbor two genes (since nine genes were to be integrated, the fifth integration site would only harbor one gene, and therefore had no use for such a promoter system), by combining promoters based on size, such that the promoter transcribing in reverse was always shorter than its non-reverse counterpart. This design would facilitate the construction of the dual promoters, as well as make sure that all four were roughly the same size.

Figure 2. Illustration of how genes are transcribed under a dual promoter. In this image, green regions represent promoters, orange regions represent genes, dark blue regions represent genetic context, i.e. genomic DNA or plasmid backbone, and light blue regions represent terminator regions that are homologous to a certain integration site in the S. cerevisiae genome.

To construct the dual promoters, the existing promoters were first to be extracted from genomic S. cerevisiae DNA by PCR amplification. The primers to be used for this PCR were designed with a region of nonsense DNA upstream of the actual promoter region, which would allow us to combine any two promoters that were extracted, using fusion PCR, since they all would have matching overhangs.

Experimental Design

The experimental design is a vital part of any project containing lab work. The experiments of this project can be divided into two categories, which are explained in detail below: construction of BioBricks and evaluation of BioBricks. Some additional experiments were also performed, including the cultivation of yeast cells in the presence of varying concentrations of PCB, to ensure that PCB is not toxic to yeast, as we found no information regarding this in the literature. The PCB concentrations used during these experiments ranged from 10 times lower than what is found in local waters to 10,000 times the levels that are deemed toxic to humans.

Construction of Composite BioBricks

The main idea behind the experimental design was to use the EasyClone marker-free integration system, for the final insertions of the BioBricks into the genome of S. cerevisiae. This is a system that consists of a set of plasmid vectors, into which genes can easily be inserted and cut out again. These can then be flanked by DNA regions that are homologous to specifically chosen integration sites in the S. cerevisiae genome, as well as a set of gRNA plasmids that encode gRNA corresponding to those integration sites, such that CRISPR/Cas9 can be used to cut those sites and the gene(s) of interest is provided as repair fragment [9]. Therefore, the composite bricks were each assigned to an integration site, and thereby a vector to be assembled into, prior to their assembly. These are displayed in Table 1.

Table 1.Genes and their corresponding integration sites in S. cerevisiae.
CRISPR site from EasyClone system Gene(s) to be integrated
XI-3 pcbA5
X-4 bphA1+bphA2
XI-3 bphA3+bphA4
XI-3 bphB+bphC
XI-3 bphD+bphK

Once this had been decided, the experimental pipeline used for the construction of the BioBricks was started. The general experimental design used for the construction of the BioBricks is illustrated in Figure 3.

Figure 3. Overview of the experimental design for constructing the BioBricks. In this image, green regions represent promoters, orange regions represent genes, dark blue regions represent genetic context, i.e. genomic DNA or plasmid backbone, and light blue regions represent terminator regions that are homologous to a certain integration site in the S. cerevisiae genome (exception for A where light blue represents nonsense DNA overhang used to fuse promoters). A. Promoters are PCR amplified from the genome using primers with overhangs, and then combined into bi-directional dual promoters using fusion PCR. B. Genes are PCR amplified using primers with overhangs to both the backbone terminator region and the promoter respectively. C. EasyClone vector is cut by restriction digestion with SbaI. D. Genes, backbone and dual promoter are combined using Gibson assembly.

As can be observed in Figure 3A, the fist thing in the experiment flow was to assemble the four dual promoters. As described before, the promoters were extracted, from genomic S. cerevisiae DNA, through PCR amplification using forward primers with specific overhangs, and the promoters were then combined using fusion PCR. The genes were PCR amplified according to Figure 3B, using both forward and reverse primers with specific overhangs. The selected EasyClone backbone for each BioBrick was acquired and linearized through restriction digestion with SbaI, according to Figure 3C. Finally, as can be seen in Figure 3D, four-part Gibson assembly was used to combine the fragments into the desired plasmid.

After this, the plasmids were transformed into E. coli. The EasyClone backbones contain an ampicillin resistance gene, and therefore the cells were grown on LB/Amp plates, to select for successful clones [9]. These were then grown in liquid media and the plasmids were extracted. The fragments containing the BioBricks and the homologous regions were cut out, through restriction digestion with XbaI, and transformed into S. cerevisiae cells, together with the corresponding gRNA plasmid. This is illustrated in Figure 4.

Figure 4. Overview of the EasyClone system, used to integrate to BioBricks into the genome of S. cerevisiae using CRISPR/Cas9. In this image, green regions represent promoters, orange regions represent genes, dark blue regions represent genetic context, i.e. genomic DNA or plasmid backbone, and light blue regions represent terminator regions that are homologous to a certain integration site in the S. cerevisiae genome.

The gRNA plasmids contain a nourseothricin resistance gene, and therefore the yeast cells were grown on selective plates after transformation. To screen for clones where the integration of the genes had been successful colony PCR was used.

Evaluation of BioBricks

The BioBricks were to be evaluated in different ways. To test whether the genes were able to be expressed, the variants of our BioBricks containing tags were used, with the expression being measured through Western Blot experiments. To test the capacity of our final strain to degrade PCB, the intention was to use GC/MS measurements, since literature had indicated that this method can be used to measure PCB concentrations [10]. The yeast would be cultivated in a medium containing a PCB concentration based on the limit of detection of the GC/MS instrument. Afterwards, two samples would be collected and analyzed, one being just the medium and one being the supernatant after lysing the cells. The PCB concentrations in these would then be measured and compared against a reference sample, containing the original PCB concentration.

Further Reading

Get a more easily accessible overview of our project in Project Description, read more about the details of the different Parts that constitute the project, check out the actual lab work we carried out documented in Notebook, or explore the simulated degradation pathways in our Modeling.

References

  1. Garrido-Sanz D, Manzano J, Martín M, Redondo-Nieto M, Rivilla R. Metagenomic analysis of a biphenyl-degrading soil bacterial consortium reveals the metabolic roles of specific populations. Frontiers in Microbiology. 2018 Feb 15;9:232.
  2. Stella T, Covino S, Čvančarová M, Filipová A, Petruccioli M, D’Annibale A, Cajthaml T. Bioremediation of long-term PCB-contaminated soil by white-rot fungi. Journal of hazardous materials. 2017 Feb 15;324:701-10.
  3. Čvančarová M, Křesinová Z, Filipová A, Covino S, Cajthaml T. Biodegradation of PCBs by ligninolytic fungi and characterization of the degradation products. Chemosphere. 2012 Sep 1;88(11):1317-23.
  4. Cajthaml T. Biodegradation of endocrine‐disrupting compounds by ligninolytic fungi: mechanisms involved in the degradation. Environmental microbiology. 2015 Dec;17(12):4822-34.
  5. Agulló L, Pieper DH, Seeger M. Genetics and biochemistry of biphenyl and PCB biodegradation. Aerobic Utilization of Hydrocarbons, Oils, and Lipids. 2019:595-622.
  6. Hofer B, Backhaus S, Timmis KN. The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes. Gene. 1994 Jun 24;144(1):9-16.
  7. Duinker JC, Boon JP. PCB congeners in the marine environment—a review. InOrganic micropollutants in the aquatic environment 1986 (pp. 187-205). Springer, Dordrecht.
  8. Bedard DL. PCB dechlorinases revealed at last. Proceedings of the National Academy of Sciences. 2014 Aug 19;111(33):11919-20.
  9. Jensen NB, Strucko T, Kildegaard KR, David F, Maury J, Mortensen UH, Forster J, Nielsen J, Borodina I. EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae. FEMS yeast research. 2014 Mar 1;14(2):238-48.
  10. Večeřa Z, Bartošíková A, Sklenska J, Mikuška P. A large volume injection procedure for GC-MS determination of PAHs and PCBs. Chromatographia. 2005 Feb 1;61(3-4):197-200.