Results

The aim of our project was to integrate nine genes into a single strain of Saccharomyces cerevisiae, enabling it to degrade PCBs. While that was not achieved, we did create strains with one or two tagged genes successfully integrated. The purpose of the tags was to test the expression levels of the genes in yeast. When the expression levels were tested with western blot, we could only observe positive results for one of the genes, bphA1. This result was later confirmed through sequencing, the results of which confirmed that only the plasmid construct containing bphA1 and bphA2 was correctly assembled. In addition, we wanted to investigate how well yeast survives in the presence of PCBs, and therefore performed experiments where S. cerevisiae was cultivated in media containing different concentrations of PCBs. The results indicated that yeast is tolerant towards PCBs, and grows well even at high concentrations. The results are described in more detail below.

Construction of Yeast Strains

The nine genes were assembled into five plasmids, here labeled P0, P1, P2, P3 and P4, with P1-P4 containing two genes expressed under a dual promoter, according to what is described on the project design page. More specific information about which genes were contained in each plasmid construct is presented in Table 1.

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**Table 1.** *Genes contained in each of the five plasmid constructs.*
Plasmid	Gene(s)
P0	pcbA5
P1	bphA1, bphA2
P2	bphA3, bphA4
P3	bphB, bphC
P0	bphD, bphK

In addition, a variant of each plasmid was also constructed, using genes which had either a His- or a FLAG-tag added before the stop codon. The results from the verification of the plasmids to the verification of yeast transformations are presented below.

Verification of Non-Tagged Plasmids

After constructing the plasmids, through Gibson Assembly and subsequently transforming them into Escherichia coli, successful clones were verified through colony PCR. Figure 1 shows the results after gel electrophoresis of the PCR products of P0, P1 and P2.

**Figure 1.** *Gel electrophoresis results from colony PCR of P0, P1 and P2. Every well contains a sample from a different colony.*

In Figure 1, two bands can be observed that correspond to the expected length of the P0 insert, which is about 1.5 kb. Furthermore, the figure shows that the colony PCR was unsuccessful for P1 and P2, since the gel only contains very short bands for their respective samples. Because the colony PCR was unsuccessful, a restriction digestion was performed to verify that the plasmids had been correctly assembled.

Verification of Plasmids Through Restriction Digestion

Two enzymes were selected for restriction digestion of each plasmid, with the aim of verifying their lengths. The enzymes selected are displayed in Table 2.

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**Table 2.** *Enzymes used for restriction digestion verification of the plasmid constructs.*
Plasmid	Enzyme 1	Enzyme 2
P0	AanI	NotI
P1	AanI	NotI
P2	AanI	NotI
P3	SacI	NotI
P0	BcuI	NotI

The expected lengths of the fragments that would be produced by each plasmid, after digestion with the enzymes listed in Table 2, are displayed in Figure 2.

**Figure 2.** *Expected fragment lengths from restriction digestion of plasmids 0-4. The plasmids are ordered numerically, with P0 being labeled 1, P1 labeled 2 and so on.*

After digestion, gel electrophoresis was performed to verify the fragment lengths. The resulting fragments are displayed in Figure 3.

**Figure 3.** *Results of plasmid verification of P0, P1 and P2 with the enzymes AanI and NotI. For each plasmid several samples are displayed, representing different colonies.*

While observing Figure 2 it can be seen that for P0, five fragments were expected, where two have lengths between 2 kb and 3 kb, and the remaining three are around 1 kb, 0.65 kb and 0.4 kb long respectively. These can be seen on the gel in Figure 3, for both of the colonies, containing P0, that were tested.

Similarly, restriction digestion of P1 with the enzymes used should produce three bands with lengths between 2 kb and 3 kb, in addition to four bands with lengths between 0.5 kb and 0.1 kb. These can be seen in Figure 3, with the longer bands appearing more clearly. The shorter bands are faint but visible for colony 1.5.

Finally, five bands were expected for P2, one close to 4 kb in length, one between 2 kb and 3 kb in length, one between 0.65 kb and 0.85 kb in length and two final bands showing lengths around 0.4 kb. Figure 3 shows that P2 has one band between 2 kb and 3 kb, and that the shorter bands are present for colonies 2.1 and 2.5. The longest band, at 4 kb, could have merged with the band with a length around 3 kb, since the bands are not very well separated on the gel. These results were deemed acceptable, and for further experiments, cells from colonies 0.5, 1.1 and 2.5 respectively were used.

Figure 4 shows an image of the gel used to visualize the results of the restriction digestion verification for P3 and P4.

**Figure 4.** *Results of plasmid verification of P3 and P4, with the enzymes SacI and NotI, and BcuI and Not I respectively.*

By observing Figure 2 it can be seen that both P3 and P4 should produce fragments around 3 kb, 1 kb and 0 kb in size. This corresponds well to what can be seen in Figure 4, which was interpreted as proof that the plasmids had been correctly assembled.

Verification of Tagged Plasmids

Following the same experimental procedure as previously described, colony PCR was first attempted with the goal of verifying the correct assembly of the plasmids containing genes with tags. These plasmids will henceforth be referred to as PT0, PT1, PT2, PT3, and PT4, with the genetic content of each plasmid corresponding to what was previously described for the original plasmids, except for the addition of tags. The results of these PCRs, after gel electrophoresis, are shown in Figure 5.

**Figure 5.** Results of gel electrophoresis of cells containing plasmids with tags. Above the wells, the first number denotes the plasmid number and the second number denotes the colony number. To the left, results for PT0 and PT1 are shown. To the right, results for PT1, PT2, PT3 and PT4 are shown.

From Figure 5 it can be observed that the colony PCR was successful for samples of PT0 and PT2, where the expected result would be a single band, at around 1.5 kb and 0.5 kb in length respectively. For the other samples, it can be seen that there are only primer clouds or multiple bands present. From these results PT0 and PT2 were considered verified for colonies 0.2, 0.5, 0.7, 0.8, 2.1 and 2.5. Due to the difficulties in verifying the remaining plasmids, restriction digestion was performed with the aim of verifying PT1, PT3 and PT4. The results from this verification attempt are presented in Figure 6.

**Figure 6.** *Results of gel electrophoresis of the digested samples of PT1, PT3 and PT4.*

The expected results for PT1 were five bands, three of them having lengths in the range between 2 kb and 3 kb, and the remaining two having lengths close to 0.4 kb and 0.7 kb respectively. Figure 6 shows these bands for the sample from colony 1.6. PT3 and PT4 were expected to show similar results to one another, both producing three bands with lengths around 3kb, 0.5 kb and 0.1 kb respectively. These bands can once again be clearly observed in Figure 6, for all PT3 and PT4 samples.

Yeast Transformation Verification

After attempting to integrate the composite BioBricks into the genome of S. cerevisiae, colony PCR was performed to verify successful integration. Figure 7 displays the results from colony a PCR of clones containing the inserts from PT0 and PT1 described above.

**Figure 7.** Results of gel electrophoresis from colony PCR products of tagged inserts 0 and 1, after integration into the genome of S. cerevisiae. Each well is labeled after the colony that the sample has been collected from.

The expected length of the fragment produced from insert 0 was around 0.7 kb. In Figure 7, this can be clearly observed for all samples containing this insert, verifying a successful integration. For insert 1, the expected fragment would instead have a length around 0.8 kb, which can be observed for sample 1.1, in Figure 7. The corresponding results for clones containing the inserts from PT2 and PT3 are displayed in Figure 8.

**Figure 8.** Results of gel electrophoresis from colony PCR products of tagged inserts 2 and 3, after integration into the genome of S. cerevisiae. Each well is labeled after the colony that the sample has been collected from.

Positive results for inserts two and three should display bands of lengths close to 0.4 kb. It can be observed in Figure 8 that all samples corresponding to insert 2 show faint bands of around 0.5 kb in length, while also displaying prominent primer clouds. This implies that the primers did not bind efficiently to the template, though the results for insert 2 were still deemed to be positive by the team. Furthermore, the samples corresponding to insert 3 show bands around 0.4 kb in length as well. In some instances, two bands can be observed, while the expected result is a single one. Because it was difficult to obtain good results from the colony PCR, due to lack of time and efficiency regarding primer annealing, this result was considered tolerable, as the inserts would be investigated further through sequencing and western blot experiments. The results from the final insert, PT4, are displayed in Figure 9, together with some additional samples containing PT3.

**Figure 9.** Results of gel electrophoresis from colony PCR products of tagged inserts 2 and 3, after integration into the genome of S. cerevisiae. Each well is labeled after the colony that the sample has been collected from.

Figure 9 shows no bands for samples containing insert 3. However, the figure does display bands for samples containing insert 4, with lengths around 0.7kb. This is most clearly seen for samples 4.5 and 4.6. As this was the expected result, insert 4 was also considered verified based on this.

While the idea for the BioBricks containing tags was to integrate each construct into a separate strain and test their expression in isolation, the same was not true for the non-tagged variants. The desire was to construct a strain of S. cerevisiae which contained all nine genes from the pathway. Thereby, all constructs would need to be integrated into the same cells. The first step towards achieving this was to attempt the simultaneous integration of inserts 2, 3 and 4 into the yeast genome. This was possible by using a plasmid that expressed three gRNAs at once, corresponding to three separate integration sites in the genome. After the transformation, colony PCR was performed to verify the successful integration of the three inserts, with separate primer pairs being used for each insert. The results are displayed in Figure 10.

**Figure 10.** Results of gel electrophoresis from colony PCR products of inserts 2, 3 and 4, after simultaneous integration into the genome of S. cerevisiae. Different primers had been used for the samples in each well.

The results shown in Figure 10 indicate that all of the inserts had been successfully integrated. In each primer pair, one of the primers attaches to the genomic region, right outside of the integration site, and the other attaches at the end of the insert. Therefore, if the integration was successful, one short fragment would be produced, which corresponds to what can be seen in Figure 10 for all samples except number 2. The remaining bands were expected to be of lengths 0.7 kb (1), 0.6 kb (2), 1.1 kb (4) 0.9 kb (5) and 0.8 kb (6) respectively, and since those bands are clearly shown, the integration was considered successful.

Concerning the continued development of the strain, containing inserts 2, 3 and 4, it did not go further, due to insert 1 not being able to be successfully integrated. As an alternative, single integrations of the remaining inserts, into separate strains, were also attempted. The idea was that, if the integrations were successful, the cells could be cultivated together, to produce the desired result. As before, colony PCR was used to verify the integration, and the results are displayed in Figure 11.

**Figure 11.** *Results of gel electrophoresis from colony PCR products of all inserts, after integration into the genome of S. cerevisiae. Wells are labeled based on the insert they contain.*

The results displayed in Figure 11 show that the integration was only successful for one of the inserts, that of P0. However, this would turn out to be the best possible outcome. Due to our efforts in human practices, the importance of the gene pcbA5, which was contained within P0, was emphasized above the remaining pathway. For this reason, coupled with the results of modeling a strain containing only the P0 insert, this strain was considered fit for further trials. The idea was to use GC/MS to measure the dechlorinating abilities of this strain. However, we unfortunately failed to develop a method, able to measure PCBs, in time. Therefore, the dechlorinating capabilities of this strain remain unknown, providing the base for a possible future project.

Western Blot Verification

Western blot was carried out to verify the expression of every gene in isolation. In each construct, the genes on either side of the dual promoter had different tags, either His- or FLAG-tag. In preparation of the western blot, the proteins were extracted and linearized. Figure 12 shows the tagged membrane, with proteins from all tagged strains, together with wild type and positive control.

**Figure 12.** *Results of western blot of His-tagged proteins. From left to right the samples, together with their His-tagged genes are WT, WT, 1 (bphA1), ladder, 2 (bphA4), 3 (bphC), 4 (bphD), positive control.*

As can be seen in Figure 12, four samples, including the wild type, have three or four bands with bound His-tags. Since the bands are also shown for the wild type samples, they can be concluded to not be the proteins produced by our integrated genes. The positive control is clearly visible, which ensures that the blot worked as intended. One band, not observed for any other samples, is present for sample 1. This band is of the same size as the BphA1 protein (51.6 kDa). Samples 2 and 4 show no bands, which could be a result of too low protein concentrations, loaded onto the gel. The ladder is only slightly visible, likely a result of the volume being loaded onto the gel being too small. There is a crack in the bands from sample 3 due to the fact that there was a crack in the gel when transferring the proteins to the blot. It is not to be confused as two separate samples.

Figure 13 shows the results of western blot with the FLAG-tagged proteins.

**Figure 13.** Results of western blot of FLAG-tagged proteins. From left to right, the samples, together with their FLAG-tagged genes, are WT, 0 (pcbA5), 1 (bphA2), ladder, ladder, 2 (bphA3), 3 (bphB), 4 (bphK), positive control.

As can be observed in Figure 13, no proteins can be seen in any of the samples that are not also present in the wild type. In addition, on this blot it can be observed that samples 2 and 4 have lower concentrations of proteins. The positive control is visible, if only slightly, in the rightmost side of the membrane.

Sequencing of DNA

The tagged variants of the gene-promoter constructs present in the plasmids were sequenced. The results showed that the plasmid construct containing bphA1 and bphA2 had the correct sequence, while the remaining constructs had been incorrectly assembled. These results further support the positive results from the western blot seen in Figure 12.

Effects of PCBs on the Growth of S. cerevisiae

To investigate whether PCBs are detrimental to the growth of yeast, the two wild type strains of S. cerevisiae, used during the project, were cultivated in media containing different concentrations of PCBs and their growth were observed over time. For reference, the IMX585 strain was transformed with the genes bphA1 and bphA2, whilst the 2CENPK 113-11C strain was used for integration of genes pcbA5, bphA3, bphA4, bphB, bphC, bphD and bphK. Each concentration was made with three replicates. Figure 14 shows the mean of the resulting growth profiles, of the strain IMX585, in six different concentrations of PCBs.

**Figure 14.** *Growth of the S. cerevisiae strain IMX585 in six different PCB concentration. Each growth profile is a mean of three replicates.*

It can be observed from Figure 14 that the IMX585 strain incubated with the lower concentrations of PCBs (0.1*10-6 - 1000*10-6 μg/μl) grows similarly to the control, growing without PCBs. The cells growing in media with higher concentrations of PCB (10 000*10-6 and 20 000*10-6 μg/μl) displayed a slightly shorter exponential growth phase, resulting in a lower OD of cells than the corresponding value for the cells growing in lower concentrations of PCBs. However, the plot clearly shows that the PCBs were not lethal to the cells and that the growth profiles of the cells subjected to high concentrations of PCBs do not substantially differ from the profiles of the control. The corresponding growth profiles for the strain CENPK 113-11C are displayed in Figure 15.

**Figure 15.** *Growth of the S. cerevisiae strain CENPK 113-11C in six different concentrations of PCBs. Each growth profile is a mean of three replicates.*

Figure 15 indicates that the cells growing in the highest concentrations of PCBs (10 000*10-6 and 20 000*10-6 μg/μl) have shorter exponential growth phases than the cells in lower concentrations of PCBs. It is clear the cells do not die from PCBs, but rather grow slower in worse conditions, with higher concentrations of PCBs. However, it should be noted that the profiles do display mean values of replicates, so if something adverse happened to one of the replicates that mean would be clearly affected.

Team:Chalmers-Gothenburg/Results