• Home
• Team
  • Biographies
  • Facilitators
  • The University
  • Official Profile
• Project
  • Overview
  • Promoter
  • Reporter
  • Chassis
  • Prototype
  • Data Page
  • Accomplishments
  • Future Directions
  • References
• Parts
  • Parts Submitted
  • Attributions
• Notebook
  • Post-Regionals
  • Journal
  • Protocols
  • Safety
• Outreach
  • Human Practices
  • Conferences
  • Follow Us!
• Sponsors
  • Sponsors
  • Acknowledgements
• iGEM

• Overview
• Promoter Project
  • Sensory Element Fishing
  • Bioinformatics and RT-PCR
• Reporter Project
  • Preliminary Testing
  • Characterization
  • Optimization
• Chassis Project
• Microalgae Tools
• Pseudomonas Tools
• Prototype
• Accomplishments
• Data Page
• Future Directions
• References

Our team set out to identify a novel responsive element towards naphthenic acid class compounds. While numerous studies have begun to identify species of bacteria which can survive and/or degrade NA’s the pathways of degradation have not been characterized. Therefore this goal required that our team design and implement novel methods in tailing ponds biological systems which may detect and potentially degrade these naphthenic acids. We used two main methods for identification of a promoter. In the first, We developed a naphthenic acid biotinylation/immunoprecipitation technique which allows for identification of proteins and protein complexes which may specifically interact with this class of compounds. Our team developed an optimized protocol for the biotinylation of cyclohexane pentanoic acid, and a protocol for the immunoprecipitation of Pseudomonas lysates using commercially available streptavidin magnetic beads.

Rationale

The sensory elements which we decided to focus our attention on were proteins which dock to DNA. Transcription factors which bind to specific elements of a gene known as the promoter region have been well characterized in other metabolic systems to respond to metabolites in the cell and change the level of transcription of particular metabolic response genes. A common example of one of these systems is the lac operon which has been harnessed as a selection system already in the registry. Identifying a system such as this for naphthenic acids, if one exists, would allow for a specific response to be generated to many different kinds of naphthenic acids. Our hypothesis requires that the organisms we use respond specifically to naphthenic acids and result in specific upregulation of metabolic genes with little background effect in the cell.

Napthenic Acid Degrading Organisms To Be Used

Due to the diversity and ambiguity of microbial species in the tailings ponds, we chose to focus our efforts for our promoter search on two species of Pseudomonas and a species of microalgae Dunaliella tertiolecta (please see our chassis section for more information on these species). All of these organisms have been shown to degrade naphthenic acids, however little work has been done in characterizing the degradation pathways of both species.

Method Design

Because we plan to screen both bacterial and microalgae screen, it was vital that our screening technique be robust and efficient in a variety of cellular systems. It was also important that it selectively targeted protein elements which were specific for naphthenic acids and did not result in unwanted background. A common technique used in similar application including protein biochemistry is immunoprecipitation (IP) for the identification of protein interactors against a particular bait protein. An antibody is incubated with the lysate of the organism of interest. The target of this antibody and any other interacting proteins of that target can be isolated using beads that are conjugated to the antibody. Isolation of the target protein along with its interactors allows for their identification through mass spectrometry (MS), a routinely used technique to identify proteins. Great successes has been produced in identifying novel pathways with this technique in a variety of organisms. figure

In a similar manner, we designed a strategy to use a naphthenic acid in a similar manner as an antibody for a target protein. This however, required that we develop a system to conjugate our naphthenic acids to a bead platform similar to the commercially available antibodies used in traditional IP. To accomplish this, we designed a protocol to react the conserved carboxylic acid of the naphthenic acid cyclohexanepentanoic acid (CHPA) (see Naphthenic Acid section) with a commercially available biotin-amine derivative. By reacting the CHPA with this biotin-amine in the presence of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) the carboxylic acid and primary amine can be reacted to an amide linkage under low pH conditions. This form of biotinylationr eaction is commonly performed with DNA and protein side chains and numerous suppliers have kits designed for these applications. However, the biotinylation of small molecules is rare and techniques involving hydrophobic compounds such as CHPA are not found in the literature. This required that we generate a novel protocol for biotinylating CHPA along with other naphthenic acid compounds. Figure – just the overall reaction

Once our naphthenic acid has become biotinylated, the biotin group will strongly interact with a protein known as streptavidin. The biotin/streptavidin interaction is one of the strongest characterized interactions discovered (Kd=1- to the -15M) and is commonly linked to magnetic beads allowing for our biotinylated naphthenic acid to be used in similar protocols as in immunoprecipitation. In theory, proteins which interact with the cyclic ring structure of cyclohexane pentnaoic acid can be isolated using our biotinylated CHPA/strepatividin beads platform and identified. Finally mass spectrometry can be used to determine the identity of these regulatory elements. Figure overall process compared to antibody IP.

Biotinylation

The biotinylation of molecules containing carboylic acid groups follows a well characterized reaction mechanism. In the context of our biotinylation reagents (CHPA and Biotin-PEG-amine) the reaction mechanism can be seen in Figure – reaction mechanism. The cyclohexanepentanoic acid will react with the carbodiimide (EDC) to produce an O-acylisourea intermediate activating the carboxylic acid making it an excellent leaving group. This requires a low pH environment to deprotonate the carboxylic acid. From this intermediate the desired product can be produced via direct attack of the negatively charged carboxylic acid with the amine group of our biotinylated compound producing a urea based compound as a by-product. Further reaction of the O-acylisourea intermediate with cyclohexanepentanoic acid may result in an acid anhydride which further reacts to produce the biotinylated compound of interest. The main byproduct of the reaction involves the rearrangement of the O-acylisourea to a stable N-acylurea. Common solutions to reduce the reaction towards this product involves the use of low-dielectric constant solvents such as dichloromethane. Due to the hydrophobic nature of cyclohexanepentanoic acid, aqueous low pH solutions will cause precidpitation of this reaction substrate, therefore, the use of other solvents such as dimethylsulfoxide need to be used. The final reaction solution used in all biotinylation experiments used 9% DCM/ 18% ddH2O / 73% DMSO. Typical reactions run overnight at room temperature. (Please see our protocols page for more information on the biotinylation experiment).

Biotinylation Confirmation

In order to confirm the biotinylation reaction is occurring in the unique conditions for our naphthenic acid standard, cyclohexanepentanoic acid, reverse phase High Performance Liquid Chromatography (HPLC) was used. A C18 analytical column from dionex was used on a Dionex Find out lisas stuff, using a protocol adapted from a Dionex provided complex naphthenic acid solutiosn protocol (please see our protocols section for more information).

To establish a set of control conditions for this data analysis, DMSO and DCM were individually injected onto the column and a read out at 210 nm on a UV/VIS spectrophotometer over a 60 min. time course figure – blank dmso/dcm.

Due to the large peak produced by both of these solvents, it was thought that identification of potential aqueous solutions which elute between 3-7 min. of the program may be masked by the large peak produced during elution of DMSO or DCM. Therefore to examine the peaks for the reactants of the solution (biotin, and EDAC) were examined in water solutions. CHPA was diluted in 80% DMSO. Biotin, EDAC, and CHPA were loaded onto the column at 25 uL of 100 mM, 500 mM, and 2mg/mL stock solutions Figure – biotin, edac, chpa

Due to the retention time of biotin and EDAC being within the rentention time of DMSO, there presence was difficult to detect during the biotinylation reaction. A reaction was set up in 80% DMSO/ 20% ddH2O to test the efficiency of reaction. A control and reaction was set up as written in the protocols section of the wiki, and incubated O/N at room temperature. Figure – dmso control and reaction zoom in

Four peaks were observed in the reaction dataset which was not observed in the control with retention times of 15 min. 19 min. 20 min. and 27 min. The 27 min. peak was found to correspond to the CHPA control. Therefore the remaining three unique peaks were determined to show that the reaction was progressing. Independent HPLC analysis showed that after 24 hours the reaction had gone to completion and there was no difference in peak sizes observed (data not shown). In order to further optimize this reaction, the reaction was repeated with and without the use of 9% DCM, in the same conditions as before. Figure reaction DMSO, reaction DCM

Comparison of the two datasets shows an increase in the three reaction peaks with relatively no change in the CHPA peak, suggesting that dichloromethane’s low dielectric constant increases the relative rate of reaction. Finally, to confirm the identity of our biotinylated product, peak fractions were collected for a potential biotin peak (~9 min.), the peak at 19 min. and the second peak at 20 min. The expected masses of biotin control and the expected products are as follows:

• Biotin = 374.50 Da
• Biotin-CHPA = 540.76 Da
• N-acylurea = 339.52 Da
• EDAC-Urea = 173.26 Da

MS analysis identified the control Biotin at the correct size as determined by the literature. The peak at 19 min. corresponds to the size of Biotin-CHPA plus a sodium ion (22.99 Da) which is found in high quanitites in the methanol used in electrospray which may be responsible for the higher peak size identified. Finally, the peak at 20 min. corresponded to the N-acylurea, the by-product of the reaction. This was an interesting result as it suggests that the DCM provided in the solution (as shown in Figure reaction DMSO, reaction DCM) did not decrease the relative amount of byproduct to biotinylated-CHPA, but the overall use of biotin in the solution, perhaps through increasing the degree of intermediate formation during the first steps of the reaction.

From the electrospray data, the peak at a retention time of 19 min. was determined to be the biotinylated product.

Immunoprecipitation and Future Directions

With the success of our biotinylation reaction, our team attempted to select for our biotinylated compound by directly incubating the biotin reaction solution with streptavidin coated magnetic beads. After reacting a biotinylation solution with 50 ug of streptavidin coated beads at 4C for 2 hours, it was identified through HPLC analysis that there was no binding of the biotin-CHPA product to the streptavidin (data not shown) likely due to the high concentration of DMSO and DCM in the solution. In order to produce a more favorable solution for this binding to occur, the reaction solution was diluted from 200 uL to a volume of 2 mL using 50 mM Tris pH 7.5 and again the beads were allowed to incubate. Once again no binding was observed when both solutions were ran using HPLC analysis. Figure streptavidin binding

No observed difference in peak height could be identified for the 19 min. peak suggesting that the biotin was not binding to the streptavidin beads. Furthermore, performing an IP using beads incubated with this reaction solution did not result in any specific bands (data not shown). This was likely due to the precipitation of the CHPA when the Tris pH 7.5 solution was added to the reaction mix. In order to successfully bind the biotinylated product to the beads, it may be required to increase the pH of the solution to 8-8.5 to ensure the CHPA will remain in solution. Unfortunately due to time constraints the University of Calgary team has not had sufficient time to complete these experiments.

In the future we hope to continue optimizing the streptavidin bead/biotinylation binding in order to perform the IP based experiments explained earlier in this document. By modifying the pH of the solution, or by attempting to slowly titrate in our reaction solution into an aqueous bead solution, our dilemma will hopefully be solved.

Conclusions

A novel system for biotinylating cyclohexanepentanoic acid as well as other model naphthenic acids has been identified and confirmed using HPLC and MS analysis. While further optmization of the protocols is required before performing IP based experiments, this technique has provided the first steps towards a novel system to detect and isolate proteins involved in the naphthenic acid detection pathways of Pseudomonas and microalgae.