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From 2012.igem.org

Week 1 (May 1-4)

Week 2 (May 7-11)

Decarboxylation

For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's "PetroBrick" (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (ADC). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).

Denitrification

In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the car operon, CarA (CarAaAbAd), CarB (CarBaBb), and CarC. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the ant operon, antA, B, and C, yielding cathecol while releasing CO2 and NH3. The car and ant operons are both regulated by the Pant regulator which is induced by the protein, antR. CarAa also has its own promoter which is not induced by antR. We have also investigated an alternative pathway using CarA combined with an amidase (amdA) that selectively cleaves NH2 from an intermediate of the car pathway. This could bypass much of the car/ant pathway and is possibly more efficient.

We have decided to use Pseudomonas resinovorans and Rhodococcus erythropolis to amplify these genes from. CarABC and AntABC from P. resinovorans has been shown to have a wide range of nitrogen containing substrate specificity. R. erythropolis contains the amdA gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its CarABC pathway.

In addition to our research we have also been learning some of the lab techniques we will be using this summer. This includes transforming a plasmid into E. coli, plating and selecting for bacteria containing the plasmid, verifying with colony PCR, performing a mini-prep and a restriction digest.

Desulphorization

Things we did.

Ring Cleavage

This week we mainly researched aromatic ring cleaving using intra- and extradiol dioxygenases from species of Pseudomonas and Bacillus but also began literature searches on aliphatic ring cleavage done by monooxygenases.

Week 3 (May 14-18)

Decarboxylation

The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.

Ring Cleavage

In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, Thauera butanivorans, contains the genes required to activate the ring by adding a hydroxyl group. The gene is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, Acinetobacter sp. SE19, contains the genes needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.

Week 4 (May 22-25)

Decarboxylation

This week, overnight cultures were grown from the colonies prepared in Week 3. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction. A restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5.

Desulfurization

This week we devised a protocol for biobricking the hpaC and started our lab work. The PCR performed on dry hpaC containing plasmids was not successful so we have to repeat the PCR. We also need to transform E.coli with these plasmids in the next week in order to make sure we wont lose all the plasmids in case the PCR was not successful again. We ordered the substrates we are going to use. Once the substrates and the Rhodococcus strain arrive we are going to test how effectively the bacteria can desulfurize different compounds.

Denitrification

This week we reviewed the primers listed in the database and also designed some new ones. Primers for CarAa, CarAc, and CarAd were designed individually, while primers for CarBaBbC and AntABC were designed to encompass multiple genes in a sequence. These primers were designed to be used on Pseudomonas putida which we decided to use as our gene source since it was available to us as opposed to ordering Pseudomonas resinovorans. We also designed a primer for the AmdA gene from Rhodococcus erthyroplois. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative AmdA pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.

We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock Psuedomonas putida on Thursday. We also resuspended the primers for CarAa, CarAc, CarAd, AntA, AntB, and Ant C that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.

Bioinformatics/Modelling

Handy bioinformatics tools

FMM (From Metabolite to Metabolite) is a critical tool for synthetic biology. FMM can reconstruct metabolic pathways form one metabolite to the other one. http://fmm.mbc.nctu.edu.tw/

This webpage runs the software that performs the enumeration of all pathways for target compounds in KEGG. More precisely, the desired KEGG compound is submitted in order to get the map of all pathways connecting the compound to metabolites endogenous to E. coli. Optionally, the full map containing the supplements can also be requested. http://bioretrosynth.issb.genopole.fr/tools/metahype/

DNA Designer 2.0 is a Drag and drop standalone software, runs on Windos, MacOS. https://www.dna20.com/secure/order.php?page=genedesigner2

DNADesign is a Web-based application http://genedesign.thruhere.net/gd/

Modelling

Constraint-based flux analysis - modeling microorganism metabolic network and perform flux analysis.
The metabolic network is translated in to a matrix and then the simulation will calculate a pathway that optimizes the goal (max biomass or max production). The network will be reconstructed (gene/reaction can be added or deleted) to enhance the expected pathway to degrade target compounds. The media (compounds, thermo-conditions) will be manipulated to find the best condition(s) that satisfy the microorganisms to digest the target compounds and reach a good growth rate (say above threshold).

Week 5 (May 28 - June 1)

Decarboxylation

None of the subproject members were present on Monday of this week (May 28). On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent location (approx. 2300 base pairs). Based on the apparent success of the gel, the products were sent away for sequencing. Stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired.