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Desulfurization Journal

Week 1 (May 1-4)

The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrificaiton, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a biosystem capable of not only detecting but also converting naphthenic acids in what will be an economically viable solution to the remediation and recovery of tailings pond water, while removing these toxic compounds before they can have significant detrimental impact on the environment.

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.


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.


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.