Figure: Structure of the C. thermocellum CipA scaffoldin CohI9–X-DocII trimodular fragment in complex with the SdbA CohII module. The backbone ribbon representation of the complex depicts SdbA CohII in blue, DocII in green, X module in rose, and CohI9 in yellow. The calcium ions and chloride ion appear as orange and cyan spheres, respectively. The modules are identified, and the N and C termini are labelled accordingly.

(Adams, Currie, Ali, Bayer, Jia, and Smith 833-839)

Abstract

There are a plethora of enzymes that occur in the natural world which perform reactions that could be immensely useful to humans. Unfortunately, the efficiency of some of these reactions may render their applications impractical. The cellulosome scaffolding protein produced by Clostridium thermocellum has been shown to significantly increase the efficiency of cellulose degradationSOURCE!. It is possible that the scaffolding protein can be reduced in size and adapted for the cell surface of Escherichia coli. Different cohesion sites on the new cell surface display protein can also be introduced to allow attachment of desired enzymes. Future applications would include producing a collection of distinct versions of the scaffolding protein for unique arrangements and concentrations of enzymes, enabling construction of an extra-cellular assembly line for a variety of multi-enzymatic reactions. This would lay the foundation for making previously infeasible applications of enzymes possible through increased efficiency.

Inspiration

Tuberculosis is caused by bacterial infections of Mycobacterium tuberculosis. The use of antibiotics in the treatment of tuberculosis is a double-edged sword: while curing the disease in some cases, it can also cause resistant mutants to emerge. Proper treatment of tuberculosis is typically a multi-drug regimen using first line anti-TB drugs: isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin (Long 425-428). However, if the regimen is not prescribed correctly or is not followed diligently by the patient due to misinformation or financial problems, the patient's population of tubercle bacilli may contain naturally drug-resistant mutants, and those mutants can become a larger percentage of the population (Long 425-428). Tuberculosis is highly contagious and the spread of resistant mutants is causing more and more drug-resistant tuberculosis cases every year (World Health Organization).

A tuberculosis lesion within the body can contain ten million to a billion bacilli, and generally 10-1000 of those are resistant to only one of the first line anti-TB drugs (Long 425-428). During mistreatment, if more than 1% of the population is resistant to only one anti-TB drug then it is termed drug-resistant (Long 425-428). Strains of M. tuberculosis that are resistant to only two first-line anti-TB drugs are multi-drug resistant, while strains that are also resistant to at least three anti-TB drugs in total are extensive-drug resistant. Drug resistance theory, which states that drug-resistance comes from pre-existing resistant mutants that are selected for by drug pressure, is the most widely accepted explanation for why multi- and extensive-resistant tuberculosis strains are emerging. (Long 425-428). The drug pressure in the case for tuberculosis is because M. tuberculosis produces a mycolic acid (a complex fatty acid) biofilm that protects it from the host’s immune system and makes drug delivery extremely difficult (Ojha, and et al 164-174). So with the mistreatment of the disease, resistant mutants can become the majority infection and the drug selection pressure of another of the first line anti-TB drugs can cause multi- and extensive-resistant tuberculosis cases (Long 425-428).

Background

The original idea for our project was to help combat drug-resistant tuberculosis infections by starting the first steps toward creating an anti-mycobacteria microbe. This microbe would be capable of breaking down the mycolic acid biofilm around the bacilli to allow drugs and the host’s immune system to eliminate the infection. This proposal meant that standardized fatty acid degradation enzymes and an easy implementation of the degrading enzymes needed to be created. E. coli's fatty acid oxidation pathway breaks down a couple different fatty acids including long chain and complex fatty acids, which is carried out by a multi-subunit enzyme that has an alpha²beta² conformation (Binstock, Pramanik, and Schulz 492-495). This multi-enzyme pathway can be isolated and over expressed in E. coli along with a cell surface display system engineered from Clostridium thermocellum's cellulosome. C. thermocellum, among other cellulose-degrading organisms, naturally produces and utilizes a scaffolding protein known as the cellulosome. This structure has been shown to significantly increase the efficiency of the organism's cellulose-degrading enzymesSOURCE!. The structure itself is composed of a number of smaller parts listed below and illustrated in the diagram.

• Cellulose-degrading enzymes - the enzymatic subunits of the cellulosome
• Type 1 dockerin regions - attached to the cellulose-degrading enzymes
• Type 1 cohesin regions - attached to the cellulosome scaffoldin protein and bind to type 1 dockerin regions
• Cellulose binding domain - attaches to the substrate and further increases the efficiency of C. thermocellum’s cellulose degradation process
• Type 2 dockerin region - attached to cellulosome scaffoldin protein
• Type 2 cohesin region - binds to type 2 dockerin region and the S-layer binding module that anchors the cellulosome to the surface of the cell

The C. thermocellum cellulosome is a good candidate for this project for the following reasons:

• The entirety of the cellulosome is located outside of the cell. This means that substrates do not have to be taken into the cell before reactions can occur.
• The cellulosome keeps enzymes within close proximity to the cell. This would be ideal for applications in sensitive environments (i.e. within another organism).
• The cellulosome significantly increases the efficiency of C. thermocellum's cellulose-degrading enzymes. The system may do the same for other multiple enzyme processes.
• The cohesin/dockerin interactions of a given species are specific to that species; the cohesin of one species will not bind to the dockerin of another.

Project Description

Project Goals

The purpose of this project is to create a template for constructing an extracellular assembly line for a variety of multi-enzymatic reactions. This would lay the foundation for making previously unfeasible applications of enzymes possible through increased efficiency. To execute this project, the following goals must be accomplished:

• Isolate coding sequences for type one and two cohesin, CtCoh1 and CtCoh2 respectively, regions from C. thermocellum's genome
• Isolate both miniA1 and miniA2 fragment sequences from C. thermocellum's scaffoldin CipA gene
• Combine miniA1 and miniA2 by means of a single sac1 restriction enzyme site
• Combine the CtCoh2 sequence with the E. coli transmembrane protein LPP-OmpA sequence, submitted by the 2008 Warsaw iGEM team

Project Overview

There are a number of issues that will have to be addressed before the cellulosome can be used in more practical applications.

• The S-layer binding module is not compatible with gram negative bacteria like E. coli. It is unable to bind to the S-layer of E. coli due to the organism’s outer membrane.
• The cellulosome scaffoldin is coded with a larger gene (roughly 7kb long) that is more difficult to work with during PCR and in plasmids.
• The addition of a greater variety of cohesin and dockerin regions (from other organisms) would be necessary to give the user more control over how the enzymatic subunits bind to the scaffoldin.
• The attachment of enzymes of the user’s choice to the scaffoldin will require that said enzymes are made to incorporate the correct cohesin regions.

Our team will address the S-layer binding module issue by hybridizing the type 2 cohesin of C. thermocellum with the LPP-OmpA part previously submitted to the parts registry by the 2008 Warsaw iGEM team. The LPP-OmpA part is a transmembrane protein with an extracellular amino acid string that will allow us to attach the type 2 cohesin of Clostridium's sdbA anchoring module without affecting the protein's structure or functionality. The LPP-OmpA part will then act as the cellulosome's new anchoring module.

To address the difficult size of the cipA cellulosome gene, the team developed an abbreviated version of the cellulosome scaffoldin by using PCR to remove from the coding domain six of the nine cohesion regions as well as the cellulose binding domain. This was accomplished by using two sets of primers that amplified two fragments of the scaffoldin gene during PCR. The first fragment termed miniA1 codes for the type 2 dockerin and the first of the nine type 1 cohesin modules. The second fragment termed miniA2 codes for the last two cohesin modules and a signaling peptide for exocytosis. The miniA1 fragment begins with the iGEM standard prefix and ends with a blunt restriction site, sac1, while the miniA2 fragment begins with sac1 and ends with the standard iGEM suffix. During assembly the two fragments were combined at the shared blunt restriction site, producing the miniA part.

Modeling

Enzymatic modeling provided by BYU iGEM

Making Our Parts

The portion of the sdbA gene that codes for the type 2 cohesin module of the sdbA protien was PCR amplified and given the iGEM suffix and a SacI prefix. The SacI prefix matches the same restriction site on LPP-OmpA's suffix. The two were ligated together after being cut with SacI.

The portion of the cipA gene which codes for the last two type 1 cohesin modules, along with the portion that codes for the first type 1 cohesin module and type 2 dockerin module, where PCR amplified. The fragments were named miniA1 and miniA2 respectively. MiniA1 was given the iGEM prefix and a SacI suffix while miniA2 was given a SacI prefix and the iGEM suffix. These two fragments were ligated together at the blunt restriction sites and inserted into the iGEM chloramphenicol-resistant backbone.

Testing

To determine whether or not the type 2 cohesin module is successfully anchored to the surface of the cells, the team must first create a fluorescence protein/type 2 dockerin module combination. Cells will be made to express both the LPP-OmpA-CtcohII part as well as the fluorescent dockerin module. If the cells fluoresce, they will be washed to ensure that any secreted fluorescing protein is not responsible. If the cells still fluoresce, the membranes will be isolated and tested alone for fluorescence. If the part works, the membrane will fluoresce indicating that the fluorescence protein is anchored to the cell membrane.

To determine whether or not the scaffoldin module works, the same experiment will be performed but with a different hybrid. In this case, the fluorescing protein would be combined with a type 1 dockerin module. It would also be ideal if more than one color of fluorescing protein was successfully anchored to the cell membrane. This would not only show that the scaffoldin can actually anchor more than one part but would also allow more thorough characterization.

Next Steps

In the future, a library of cohesin and dockerin modules could be built for simplified assembly of customized scaffodins. To do this, the team would isolate cohesin and dockerin modules of a variety of types from a variety of cellulosome-producing species. These modules would then be combined by iGEM non-standard restriction sites flanking the gene sequence, allowing for almost complete customization to the user's needs.

The team may also attempt to test the system using a naturally occurring multiple enzyme process (possibly fatty acid oxidation). The efficiency of such a system could be measured with and without the modified scaffoldin and anchoring protein.

References

1. Adams, J.J., M.A. Currie, S. Ali, E.A. Bayer, Z. Jia, and S.P. Smith. "Insights into Higher-Order Organization

   of the Cellulosome Revealed by a Dissect-and-Build Approach: Crystal Structure of Interacting "Clostridium thermocellum" Multimodular Components." Journal of Molecular Biology. 369. (2010): 833-839. Web. 1 Oct. 2012.

2. Binstock, J. F., A. Pramanik, and H. Schulz. "Isolation of a Multi-enzyme Complex of Fatty Acid Oxidation from

   "Escherichia coli"." Biochemistry. 74.2 (1977): 492-495. Web. 29 Sep. 2012.

3. Chao, Tiffany. "Tuberculosis Becoming More Drug-Resistant Worldwide." ABC News. ABC News Medical Unit, 30 August

   2012. Web. 28 Sep 2012. <http://abcnews.go.com/Health/tuberculosis-drug-resistant-worldwide/story?id=17107153&gt;.

4. Long, Robert. "Drug-resistant tuberculosis." Canadian Medical Association Journal. 163.4 (2000): 425-428. Web. 28

   Sep. 2012. <http://www.ecmaj.ca/content/163/4/425.full.pdf&gt;.

5. Ojha, et al. "Molecular Microbiology." Growth of "Mycobacterium tuberculosis" Biofilms Containing Free Mycolic Acids

   and Harbouring Drug-tolerant Bacteria. 69.1 (2008): 164-174. Web. 29 Sep. 2012. <http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06274.x/pdf&gt;.

6. "Tuberculosis." World Health Organization. N.p., March 2012. Web. 28 Sep 2012. <http://www.who.int/mediacentre/factsheets

   /fs104/en/&gt;.

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