Team:Calgary/Notebook/Desulfurization

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Desulfurization Journal

Week 1 (May 1-4)

During this week, literature search was performed.

Week 2 (May 7-11)

Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity, desulfurization rate, and a need for a carbon source to regenerate co-factors. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.

Week 3 (May 14-18)

Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for dszA, dszB, and dszC which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists. dszD, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is encoded on the chromosome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. Rhodococcus erythropolis IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.

The 4S Desulfurization Pathway

An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the E. coli W genome. This enzyme has been shown to increase the rate of desulfurization by x amount find citation Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the R. erythropolis , while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.

Week 4 (May 22-25)

This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the hpaC gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the Rhodococcus strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper published by REFERENCE , whos team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.

Week 5 (May 28 - June 1)

Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing A, K, T and C antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against hpaC, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the E.coli cells. The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure hpaC with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the hpaC gene into the pSB1C3 vector. All the sets grew successfully. Following the above successes with hpaC, the arrival of our Rhodococcus strain afforded us the opportunity to begin investigation of the Dsz operon using the primers current in our possession. This strain is an environmental isolate that has been shown by someone to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using dszA primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C). DO WE HAVE A PICTURE

Week 6 (June 4 - June 8)

In order to confirm the hpaC biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the pSB1C3 backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with hpaC primers and with stanndard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using hpaC primers FIRST PIC(However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative hpaC biobrick. Digestions were performed on the miniprep products using EcoRI and Pst to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for hpaC)(second figure). hpaC was sent in for sequencing.

[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|500px|center]] [[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center]]

PCR reagents were prepared to re-test/confirm previous results of dszA amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. WHY DID WE PROCEED WITH THIS THAT ISNT THE RIGHT SIZE Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.

Week 7 (June 11 - June 15)

This week, we focused on amplifying dsz genes from our Rhodococcus strain for construction into biobricks. We also wanted to purify the PSB1C3-hpaC and pUC18-hpaC plasmids to replenish our current stocks. For the dsz aspect, we were able to successfully grow extra plates of Rhodococcus strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.

PSB1C3-hpaC verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (KatG-LAA, BBa_K137068)sent as a culture stab from the parts registry was initiated, with our newly identified biobricked-hpaC acting as a positive control, but the banding pattern was not very conclusive.

Week 8 (June 18 - June 22)

PCR was reattempted on Rhodoccocus that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the Rhodococcus cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/uL to 182.7ng/uL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained.

Week 9 (June 25 - June 29)

PCR was attempted to amplify the genes of the dsz operon utilising an adapted PCR protocol with purified Taq polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55C to 65C. This was assumed to be indicative of successful amplification of dszB; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine.

Week 10 (July 2-July 6)

Top 10 E.coli cells were transformed with R0011 (IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the KatG-LAA gene was most likely present.

Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg

PCR using Phusion high fidelity polymerase was carried out on dszA, dszB, and dszC in a gradient thermocycler. Amplification of non-specific bands was present for dszA and dszB, however strong banding for the desired size of the gene was observed for both (around 1500 for dszA, 1100 for dszB

Ucalgary2012 6.7.2012.dszABphusionPCR.jpg

Examining the sequences of the dszABC genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. dszA has four PstI cut sites, dszB has a PstI and a NotI and dszC has a PstI cut site. In order to eliminate cut sites, the Stratagene QuikChange mutagenesis procedure is going to be used, with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above.

Week 11 (July 9-July 13)

Following successful amplification of the dsz operon genes in the previous week, the genes were constructed into the PSB1C3 vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part J13002 (pTetR)in front of the previously biobricked hpaC was attempted. Overnight cultures were also prepared using two colonies each for J13002 and R0011 (an IPTG inducible promoter that we hope to build in front of B0034). These cultures were then miniprepped to yield the respective parts.

PICTURE

Additionally, katG-LAA was built into a PSB1C3 backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day. On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of Rhodococcus when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the Rhodococcus.

Week 12 (July 16 -July 20)

This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of BBa_J13002 with hpaC was also explored by using forward and reverse primers of R0040 (the promoter component of the composite part J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.

Week 13 (July 23 - July 27)

Mutagenic primers were redesigned after initial design was found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of R0011 into a chlor-resistant vector was necessary, so this was attempted. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then miniprepped before finally being digested with enzymes EcoRI and PstIA. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with katG-LAA constructed into a PSB1C3 backbone, as well as the hpaC+J13002 construct. katG was shown to have been successfully amplified, verifying its presence in the colonies, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either R0011 forward primers or B0034 primers were used and the overall constructs were made either on a chlor-resistant, or amp-resistant vector. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success.

Week 14 (July 30 - August 3)

Sequencing results from the previous week's constructs were available confirming the contruction of KatGLAA in a chlor-resistant backbone, however, the backbone switch of R0011 also to PSB1C3 was not successful, probably owing to the small length of the RBS. On the other hand, the construction of J13002+hpaC was finally sent in for sequencing too, pending results. Site-directed mutagenesis of the dsz operon was also initiated - dszA has four PstI cut sites. dszB has a PstI and a NotI site. dszC has two PstI cut sites. Mutagenesis was started this week to change a single base pair in these genes in a way that the cut site is eliminated but the amino acid the gene codes for stays intact. In addition another mutagenesis has to be performed in order to replace the Tyr at position 63 of dszB gene with a Phe. Ohshiro 2007 showed that this mutation increases the activity of the enzyme. The mutagenesis was started by mutating the second PstI site in dszC (PstI2) and a control plasmid containing a point mutated β-galactosidase gene was also mutated with its specific primers. Kappa Hifi kit was used for all the mutagenesis. After the PCR mutagenesis and running a part of the PCR products on the gel, amplification was observed. Then the PCR products were DpnI digested to degrade the parental DNA and they were transformed. Control PCR products were plated on an amp plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked and the β-galactosidase gene was now functional. Then O/N culture of dszC mutants were miniprepped and digested with PstI enzyme and the results were successful (Lisa gel pic).

Attempts to do all the mutations in the genes in one step using the Knight procedure failed (http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site). This procedure used Taq ligase buffer. We suspected that the reason this procedure is not working might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out the buffer that they both work best in. The result was that both enzymes (kappa polymerase and Taq ligase) work best in a buffer made of half Taq ligase buffer and half kappa polymerase buffer.

Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg
UCalgary 02.08.12 dszC psti digest mutagenesis.jpg

Week 15 (August 7 - August 10)

Sequencing results for J13002/hpaC returned negative, so a 3-way ligation method was used to retry the construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: J13002(E/S) + HpaC (X/P) + PSB1K3 (E/P). Also, the more conventional construction of J13002(S/P) + HpaC(X/P) was reattempted. Furthermore, 3-way ligations were also attempted for B0034+KatGLAA+PSB1K3, and R0011+B0034+PSB1C3, as well as the two-way contruction of just KatGLAA after the B0034. After plating these transformations, colony PCRs were carried out on selected colonies and various samples that gave an indication on the gels of being successful were used to prepare O/N cultures then subsequently miniprepped.(insert gel images!) With regards to the site-directed mutagenesis side of the experimentation, dszA-PstI1 (the first PstI cut site in dszA) , dszB-PstI and dszC(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation. Multisite mutagenesis was repeated again using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using pfu Turbo polymerase and following the Knight procedure without any modifications. No successful results were observed.

Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg

Week 16 (August 13 - August 17)

Following on from the progress in the previous week of the mutagenesis, dszB(PstI mutated)-Y63F and dszA(PstI1 mutated)-PstI3 mutagenesis were performed. The gel below shows the digest confirmation.

Ucalgary2012 15.08.2012 dszAPstI1&3 dszB Psti y63f muta diges-1.jpg

Another approach was tried to do mutagenesis with a quicker pace. In this approach, after the PCR mutagenesis, the PCR products were purified. Afterwards they were incubated with T4 PNK and ligase. After heat inactivating ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.

Sequencing results came back. dszA (PstI1 and PstI3 mutated) and dszB(PstI and Y63F mutated) were good. However dszC (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the dszC(PstI2 mutated.

dszB(PstI and Y63F mutated)-NotI and dszA(PstI1 and PstI3 mutated)-PstI4 mutagenesis were performed. The gel below shows the digest confirmation. To investigate the actual desulfurisation capability of our initial Rhodococcus source culture for the dsz operon, a desulfurization assay was prepared by innoculating various treatments of the M9 media prepared previously. Also, a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s). Elsewhere, B0034 was constructed with dszC and of the colonies that grew on the plate of the transformed products, colony PCRs were performed and the gel image gave an indication of it being successful. However, further analysis through sequencing showed that the constructions were not successful, and so the constructions were restarted.

Week 17 (August 20 - August 24)

This week, progress was made in determining the desulfurization activity of our Rhodococcus strain as measured by the sulfate release using a turbidometric assay. We encountered numerous challenges in our prescribed protocol as the concentrations that we are using to prepare out standard curve may be too dilute, or the composition of out conditioning agent may be flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through GCMS analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the dsz operon will be further investigated.

Since the dszC second mutagenesis had proven to be unsuccessful last week, the dszC(PstI2 mutated)PstI1 mutagenesis was repeated. Also dszA(PstI1,3,4 mutated) PstI2 mutagenesis was performed. dszA and dszC were sent for sequencing on Wednesday. dszB was sent for sequencing on Friday. Sequencing results of dszA and dszC were back by Friday. dszC was successful. However, dszA contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. dszB(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. J13002-dszB and B0034-dszC constructions were attempted, however they were not successful as indicated by cPCR. Constructions of J13002/hpaC were carried out and also came back negative in sequencing, however B0034/katG-LAA was sequence confirmed.

Week 18 (August 27 - August 31)

dszB sequencing results came back as successful. dszA(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also dszA(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing.

Constructions of J13002/dszB and B0034/dszC were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of dszA

At this point, all of the dsz genes have been successfully made biobrick compatible, and hpaC has been biobricked. We have also successfully constructed B0034 with katG-LAA to be used in the optimization circuit, as well as J13002/dszB and B0034/dszC.

Constructions of J04500 with hpaC, dszB, and katG-LAA were performed. As well, attempts to construct J13002/dszB/B0034/dszC as well as J13002/hpaC, B0034/dszA, and J13002/katG-LAA were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H2O2 stress). Transformations of all these constructions were carried out at the end of the week.

During this week, part of the sulfur team was also working on the transposon screen for FRED.

Week 19 (September 3- September 7)

Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of J04500/hpaC, J04500/dszB, and B0034/dszA. Sequencing was sent, and results indicated that the constructions of J04500/hpaC were successful, meaning that after many months of trying we FINALLY have a promoter in front of the hpaC gene and can proceed to test the parts functionality. Attempts to construct hpaC with the evil TetR promotor (J13002) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. J04500/dszB also worked, though this was less exciting. B0034/dszA came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of J04500/katG-LAA and J13002/dszB/B0034/dszC were reattempted, however only J04500/katG-LAA produced colonies. Confirmation digests for this part looked good, and so samples were sent for sequencing.

Part of the sulfur team continued to work on transposon screen work as well

Week 19 (September 3- September 7)