Team:CoBRA/Project

   CoBRA iGEM Project Proposal Our research is to determine whether chitinase genes in transgenic E.coli will be produced and to what degree to reduce the BSF disease in pine trees. The CoBRA iGem team will attempt to engineer a new DNA biobrick containing a specific promoter gene, a gene of interest (one of three different Class I chitinases; PgeChia1-1, PgeChia1-2, and PcChia1-1,)3 and a terminator gene. This construct will then be placed in E.coli bacteria so that this bacteria, when subjected to pine tree resins, will secrete the chitinase which will kill the Gc.

   3N. Kolosova, J. Bohlmann, and C. Breuil. "Cloning and characterization of chitinases from interior spruce and lodgepole pine."Elsevier (2014): 1-8. Print.

   Background Information For our project, the CoBRA iGEM team has decided to attempt to manage the devastation that the mountain pine beetle (Dendroctonus ponderosae) has caused to the pine trees in forests along the Rocky Mountains in Alberta and British Columbia. This beetle burrows into the bark of the lodgepole pine of these Mountains and with the help of a fungus called the Blue Stain Fungus(Grosmannia clavigera), lays its eggs in the tree. The winter then kills the beetle off, but the eggs, which are protected from the cold by the tree, survive the winter and mature to be able to start the cycle all over again. The Gc spreads its mycelium into the phloem, and then feeds on this essential structure, thus choking off the supply of glucose to the tree. Research also shows the the BSF survives the pine trees defensive resin production, by using the monoterpene chemicals as its food source.

   These processes put the tree under tremendous stress and often kills it. The Mountain Pine Beetle has always been a factor in this ecosystem, but historically, winters have been harsh and cold enough to keep the beetle population in check. With global warming beginning to produce noticeable increases in winter temperatures, more and more beetles have been able to survive, and the population has reached a level where it is capable of ravaging huge chunks of forest, not only destroying the ecosystem, but rendering the lumber unusable.

   Looking at this problem from several angles, our team decided that the best way to combat this epidemic would be by killing off the Blue Stain Fungus or Gc. One reason is that the BSF is helpful, but not essential to the MPB survival. Secondly, by focussing on the BSF, we are attempting to minimize our disruption of MPB predator populations, such as woodpeckers and other birds. Thirdly, our objective is to focus specifically on the pathogenic BSF and not destroy the symbiotic relationships of a forest fungal ecosystem.

   Chitin is a structural component of the cell wall of many pathogenic fungi including BSF. Chitinases are enzymes that hydrolyze the polymer chitin breaking it down. Extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Expression of cloned chitinase genes in transgenic plants has provided further evidence for their role in plant defense. The level of protection observed in these plants is variable and may be influenced by the specific activity of the enzyme, its localization and concentration within the cell, the characteristics of the fungal pathogen, and the nature of the host-pathogen interaction. The expression of chitinase in combination with one or several different antifungal proteins should have a greater effect on reducing disease development, given the complexities of fungal-plant cell interactions and resistance responses in plants.

  Our project goal is to determine if a cloned chitinase cDNA can be successfully expressed in transgenic E.coli. Using recombinant DNA techniques our team will create an entirely new DNA biobrick, this biobrick will placed in the pSB1C3 vector and contain a specific inducible or constitutive promoter (LacI or TetR), a specific cDNA (one of three class 1 chitinases; PgeChia1-1, PgeChia1-2 and PcChia1-1), and a stop codon or terminator gene. This construct as previously mentioned will be placed into lab grade Top 10 and k12 lab strain E.coli bacteria thus allowing these new, genetically altered bacteria to successfully produce and secrete the chitinase enzyme thus showing proof of concept. It is important to note that our engineered bacteria will not be used outside of a controlled lab setting during our for the current project.

Materials and Method:

  As our ultimate goal is to express the chitinase gene in transgenic E.coli, we followed standard recombinant DNA techniques. To begin, our specific gene of interest, the chitinase, is not available in the iGEM registry. The Chitinase genes were received from Dr. Bohlmann at UBC. The DNA needed to go through some additional changes before they were usable. First of all, these pieces contained the PstI cut site in their coding region, which could wreak havoc on our restriction digest, by cutting the Chitinase at the wrong place with the enzymes. In addition, the biobrick prefix and suffix were not present, so the Chitinase parts could not be ligased to any of our other parts.

	Pc 1-1	Pge 1-1	Pge 1-2
Before optimization
After optimization

  We optimized our DNA using BioBasics Inc. Once we received the optimized DNA we proceeded to create our new biobricks. The vector for our chitinase DNA was different than the standardized vector required by iGEM. Utilization of the iGEM protocols for DNA recombination techniques enabled our team to create transgenic E.coli DH5-alpha cells in the proper iGEM pSB1C3 backbone. At this point we then needed to identify that our transformed DH5a cells contained our correct plasmids. We performed two sets of gel tests. First we digested the J04450 cells containing our gene of interest, as well as the pSB1c3 backbone and tested using gel electrophoresis, comparing our results to a standard ladder. Next we digested our J04500 cells which was a construct consisting of our chosen promoter, RBS, gene of interest and terminator comparing the results to a ladder.

To end we submitted all six of our biobricks to iGEM. We performed all of our protocols under aseptic conditions and were conducted at room temperature unless stated otherwise.

Result

DIY Shaking Incubator: Hova-Bator Incubator Atop an Electric Plug-In Baby Swing

Failed Bacterial Transformation Plates Using NEB Top 10 E.coli May 11, 2014

Notice the odd fringe of growth around the central core. Notice the colony shape is not circular or dense in the core.

Successful Bacterial Transformation Plates Using DH5a E.coli May 12, 2014

Future goals

Idea #1
http://www.pnas.org/content/108/6/2504.short

• We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. *Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

*Terpenoid - Any of a large class of organic compounds including terpenes, diterpenes, and sesquiterpenes. They have unsaturated molecules composed of linked isoprene units, generally having the formula (C 5H 8)

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Idea #2 - Monday March 10 from Magda

Surface display: Instead of sending things to the media outside the cell, you can also stick things onto the outside of the cell to be displayed. As Magda suggested, this normally works just fine, however sometimes it can impair the function of your protein. The only way to tell for sure is to try however- usually you fuse the protein (as described for the secretion tag) to the N terminus (start) of the protein for one construct, and the c terminus (end) of the protein for a second construct, and test both to see if one works better. The part that Magda suggested has been shown to work. http://parts.igem.org/wiki/index.php/Part:BBa_K103006. An alternative would be something like this https://2012.igem.org/Team:Penn/SurfaceDisplayBBa. It won best biobrick, and was also shown to work. Chitinase APPEARS to be a monomer (meaning that the enzyme is formed from a single protein chain, and not from more than one protein associating together (Protein subunits)). I think this article brings strong support in favour of targeting the blue stain fungus with a bacteria-displayed chitinase. And the fact that the surface display part (http://parts.igem.org/Part:BBa_K811005) is available in the registry, and even won the best biobrick award (!) adds to the promise of this project. Thanks for digging these out, Lisa

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Idea #3 - Saturday March 29 from Richard Lee

Arming Trees Against Pine Beetle Invasions

Appendices

Pc Chia1-1 optimization      Project overview
Pge Chia1-1 optimization     Kolosova et al.2014 Phytochemistry
Pge Chia1-2 optimization     Stage One Report The BC Experience and Lessons for GAER Final January 2007