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Team:CoBRA/LabNotebook
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<b>Entry #40 - Sites with Lysis ideas</b></div><div style="text-align:left"><b><br /></b></div><div style="text-align:left">These sites have ideas for a way to lyse cell while leaving the protein intact</div><div style="text-align:left"><br /></div><div style="text-align:left">http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/</div></div></div></div></div></div></div></td></tr> | <b>Entry #40 - Sites with Lysis ideas</b></div><div style="text-align:left"><b><br /></b></div><div style="text-align:left">These sites have ideas for a way to lyse cell while leaving the protein intact</div><div style="text-align:left"><br /></div><div style="text-align:left">http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/</div></div></div></div></div></div></div></td></tr> | ||
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Revision as of 20:55, 17 June 2014
Entry #2: This is an excellent diagram illustrating the interactions between the mountain pine beetle, the blue-stain fungus and the pine tree. Will this help us narrow our research?
Hello Adam,
Thank you for contacting me, and your interest in collaborating with the Tria team around your iGEM competition. I have forwarded you email and contact information along to the Network Director, Dr. Janice Cooke, as well as our research team for an information they can provide. Normally I'd be happy to provide you with information, however I will be away for the next two weeks without email contact and would be unable to help at the early, and critical stages of your project development.
The interactions amongst major biological components of the MPB system (the beetle, pine tree hosts, fungal associates, and bacterial/other associates) are very complex, and you are correct that you want an in-depth perspective before deciding on the research direction you with to explore. I would encourage your team to read more about the life cycle of the beetle before settling solely on an E. coli derived solution.
If I could recommend some resources for you, the Tria project website has a series of research papers on more specific attributes of the MPB epidemic which could be useful once you team has decided on its final direction. Also, I would recommend reviewing the MPB website operated by Alberta Environment and Sustainable Resource Development who are the hands on Government organization who conduct monitoring and control activities in Alberta. Another site to review would be that of the Canadian Forest Service which provides a more Canadian perspective on the issue. Likely you have come across these as you researched the topic, but I'm still providing links below:
- Tria Project Website: http://www.thetriaproject.ca
- AESRD MPB website: http://mpb.alberta.ca/
- CFS website: http://www.nrcan.gc.ca/forests/insects-diseases/13381
I will follow up with you once I return from absence and see if there additional information I can provide. I wish you and your team the best as you choose your research topic and move ahead in a fun competition.
Cheers,
Matt
Matt Bryman
Network Manager
The NSERC TRIA Network (TRIA-Net)
Hi Stephanie,
Apologies
for the delay in responding to your email. We sent out a request to
our TRIA members to see if anyone would be interested in mentoring your
iGEM team in this project. I was disappointed that no one was available
to mentor your team on this. I am probably not the most appropriate
person to guide your group, but I would be willing to take a stab at it.
I have spent quite a lot of time thinking about synthetic biology products that TRIA would be interested in, and how we could provide guidance. My lab is not really in the business of biotechnology, so this is a stretch for me. After some thought, I think that there are two avenues that the Cochrane students could take.
The first avenue would be more aligned with TRIA research: here, your students could try to develop a system that synthesizes different mountain pine beetle pheromones or tree chemicals that can cause changes in beetle behaviour that could help in controlling the outbreak. An example of a commercially available beetle pheromone is verbenone, which is emitted by beetles to signal that an attacked tree is fully occupied, and that incoming beetles should find another tree. Similarly, maybe there is the potential to synthetically create a blend of tree chemicals that could be used as a repellent (or as a trap). I have cc’d two TRIA researchers on this email who have expertise in this area; if you wanted to follow up on these ideas, I would hope to convince them to provide some mentorship on this project.
The second avenue is quite far from TRIA research: here, your students could use mountain pine beetle killed timber (infected with the mountain pine beetle fungal associates) as a feedstock to create new products from this wood. Given that the fungus is degrading the wood, the wood composition will be different than conventional softwood, and potentially could be used to generate different extractives, such as for the biofuel industry. This would be fundamentally exciting, but is very far from what I do. I have a colleague on campus who may be able to help you on this project, if your team was interested in this avenue.
Apologies again for the delayed response. Please let me know if you would like to proceed with one of these projects (or if you’ve found another mentor), and we can decide next steps.
Best wishes,
Janice
Associate Professor
Department of Biological Sciences
CW405 Biological Sciences Building
University of Alberta
Edmonton AB T6G 2E9 CANADA
Tel: 780.492.0412
Fax: 780.492.9234
email: janice.cooke@ualberta.ca
Office: CCIS 5-108
Entry #5: Mountain Pine Beetle
http://www.nrcan.gc.ca/forests/insects-diseases/13397
This site has the scientific name of the mountain pine beetle (Dendroctonus ponderosae), as well as its' life history and how it attacks and damages the tree.
http://www.publicaffairs.ubc.ca/2011/01/24/genome-of-blue-stain-fungus-evolved-to-bypass-tree-defense-in-mountain-pine-beetle-epidemic-ubc-research/
"Now, researchers from UBC and the BC Cancer Agency’s Genome Sciences Centre have conducted a detailed genome analysis and identified genes in Grosmannia clavigera that are responsible for the fungus’s ability to bypass the lodgepole pine’s natural fungicide – and use it as a carbon source for fungal growth."
http://www.ncbi.nlm.nih.gov/pubmed/20727970
"The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids."
Entry #8: Verbenone uses
Entry #10: Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum
Scientific name: Grosmannia clavigera
Kingdom: Fungi
Phylum: Ascomycota
Class: Sordariomycetes
Order: Ophiostomatales
Family: Ophiostomataceae/p>
Genus: Grosmannia
Species: G. clavigera
http://www.pc.gc.ca/docs/v-g/dpp-mpb/sec3/dpp-mpb3a.aspx
“Mountain pine beetle carry fungus spores near their mouthparts”
“the chewing action empties the spores into the tissues of the tree”
“After the beetle introduces the fungi to the tree, *mycelia grow rapidly into the sapwood, slowing the movement of fluids and disrupting the tree’s defences against the invading beetles”
*Mycelia is the vegetative part of a fungus that grows in thread-like structures
Mycelia grows in the phloem and blocks the flow of fluids, which would block the flow of sap. This sap would trap the beetles and kill them if the fungus was not present
Guy that researched blue stain fungus genome and identified genes that are able to convert natural tree fungicide into food source:
Joerg Bohlmann (EMAILED)
Michael Smith Laboratories
Tel: 604.822.0282
Cell: 604.347.8843
E-mail: bohlmann@interchange.ubc.c
http://www.cfs.nrcan.gc.ca/bookstore_pdfs/28144.pdf
The fungus bypass the trees natural defences due to a genetic change that allows them to turn these defenses into a carbon based food source.
Mountain Pine Beetles serve as a vector for three different types of blue stain fungus, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum
Based on the fungi’s isolated location (Alberta, BC, Colorado, California, etc.) different fungal isolates immerge with varying temperature tolerances and optimal growing conditions.
“The precise role of these fungi is not known, but they are thought to help the beetle overwhelm the defences of the host tree… and provide necessary nutrition throughout the lifecycle of the beetle”
Grosmannia clavigera is the more aggressive of the three fungi on most host trees.
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)
Entry #11: Random thoughts and ideas
Female pine beetles would release pheromones (aggregation pheromone) once in the tree to attract male beetles for reproduction. They are also capable of releasing pheromones (anti-aggregation pheromones) that contradict these pheromones that give the message to other beetles that the tree is already occupied. These pheromones only have a small area around the beetle where they are actually effective. I was thinking if there is any way to take the anti-aggregation pheromone and to neutralize it with the aggregation pheromone. if we neutralize this pheromone will this stop the male beetle from finding the female beetle? Or will the time it will take for the two beetles to meet just be slowed down and give time for the trees natural defenses to kick in and kill the female beetle? Obviously this will take some more research but thought I should just write my thoughts down before they slipped away.
Entry #12: Chitinase Info.
Entry #14 February 11, 2014 Chitinase Info
Chitinases are enzymes that hydrolyze the N-acetylglucosamine polymer chitin, and they occur in diverse plant tissues over a broad range of crop and noncrop species. The enzymes may be expressed constitutively at low levels but are dramatically enhanced by numerous abiotic agents (ethylene, salicylic acid, salt solutions, ozone, UV light) and by biotic factors (fungi, bacteria, viruses, viroids, fungal cell wall components, and oligosaccharides). Different classes of plant chitinases are distinguishable by molecular, biochemical, and physicochemical criteria. Thus, plant chitinases may differ in substrate-binding characteristics, localization within the cell, and specific activities. Because chitin is a structural component of the cell wall of many phytopathogenic fungi, extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Plant chitinases have different degrees of antifungal activity to several fungi in vitro. In vivo, although rapid accumulation and high levels of chitinases (together with numerous other pathogenesis-related proteins) occur in resistant tissues expressing a hypersensitive reaction, high levels also can occur in susceptible tissues. 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. The effects of plant chitinases on nematode development in vitro and in vivo are worthy of investigation.
Entry #15 February 12, 2014 BSF Genome info
http://treephys.oxfordjournals.org/content/32/8/943.full
http://www.pnas.org/content/108/6/2504.full
Entry #16 Chitinase experiment
Entry #18 New Direction to Consider - February 18, 2014
From Dr Janice Cook
Expressing the chitinase in the bluestain fungus itself (Grosmannia clavigera would be an appropriate choice). In an ideal world, the chitinase would be secreted by the fungus into the cell wall space, and chew up the chitin. Then the fungus (particularly fungal growth) is compromised because of the degradation of the cell wall.
You want to have the chitinase turned on when the fungus is introduced into the tree, so that its growth is compromised. So maybe there is a fungal gene that is strongly upregulated by something about the tree, like monoterpenes? If Joerg's group (or Colette's group) has identified one or more of these genes (which I'm pretty sure they have), then you can identify the promoter for this gene from the G. clavigera genome sequence. This can be used to create the construct that you would use to create the transgenic fungus, which then could be introduced into the tree (or at least, for proof of concept, onto culture plates that contain the monoterpenes) and see if you can turn on the gene. If the gene gets turned on, then you can see what happens to the fungal cell wall.
From Magda Pop - in an earlier gmail - which could be what Dr Cooke is speaking to
You could use a (pinII) promoter which is induced by monoterpenes
(stress tree chemicals) to control a chi gene => when the tree gives off
monoterpenes, the (pinII) promoter is activated to produce chitinase enzyme,
which degrades the chitin from the cell wall of the fungus ***Note: the pinII
promoter might be available in Vancouver (http://www.researchgate.net/publication/6167104_Testing_of_a_heterologous_wound-_and_insect-inducible_promoter_for_functional_genomics_studies_in_conifer_defense
)
Or instead of pinII, use a stress/terpenoid-induced promoter that dr Bohlmann
recommends?
From Patrick Wu in response to Dr Cook
If we engineer the fungus directly, however, I'm not too certain if the fungus will be willing to keep those genes as it reproduces. Suicidal organisms tend not to be considered evolutionary fit, so those genes might simply disappear in one or two generations...
But we mentioned a different organism during our brainstorming session, Agrobacterium tumefaciens. This is an organism that usually infects plants and can introduce DNA into them (which is how scientists can transform algae--for cheap!). What can be done, if we can't express the chitinase in E. coli, is to instead grow Agrobacterium that can introduce the chitinase genes into the fungus. From there, the fungus can create the chitinase inside itself.
There seems to be at least a few papers on Agrobacterium being able to introduce DNA into blue stain fungus. I've attached one here.
Blue Stain Fungus Transformation.pdf
The only issue with this idea is accidental transformation of the trees along with the fungus, since the bacterium was originally a plant pathogen. That might be something to explore further... hm.
Entry #19 Building From Latest Consideration - February 19, 2014
From Dr Bohlmann
We have a small paper in press on spruce and lodgepole pine chitinases which I will send you as soon as have the pdf of the corrected page proofs. You can also look for the paper at:
http://dx.doi.org/10.1016/j.phytochem.2014.02.006. (the link does not work) I should be online within the next couple of weeks. The paper does not address your questions about engineered promoters for controlled expression, but includes some basic information on simple IPTG-inducible expression in E. coli.
From Magda in response to Dr Bohlmann
I must say I am not clear how dr. Cooke's idea would work in practical terms.
Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, (this is a standard promoter) and it is therefore used to induce protein expression where the gene is under the control of the lac operator (which chitinases are under).
IPTG, unlike allolactose, is not hydrolyzable by β-galactosidase,
its concentration therefore remains constant in an experiment.
Like allolactose, IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. But unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inducer. The concentration of IPTG therefore remains constant and the expression of lac p/o-controlled genes would not be inhibited during the experiment.
According to Dr Bohlmann, if we grow E. coli in a solution of IPTG, the bacteria will take it up and undergo transcription. If we engineered an E. coli construct with a spruce/pine tree chitinase, then that transgenic E. coli will produce the tree chitinase.
Entry #20 Clarification of Terms From Latest Consideration - February 21, 2014
A constitutive promoter is normally ON, meaning that a coding region downstream from it will be always transcribed under normal/regular conditions. For example, if you placed the coding region for the chitinase downstream from a constitutive promoter, under normal conditions of growth the bacterial cells will make the chitinase enzyme continuously (ie all the time).
Entry #21 Meeting with David and Himika - February 25, 2014
Entry #22 Origin of the chitinases - Wednesday March 8
Entry #23 Ken Raffa
Entry #24 Nucleotide/ Protein Sequences: Saturday March 8
PgeChia1-1 http://www.ncbi.nlm.nih.gov/nuccore/HM219843
PgeChia1-2 http://www.ncbi.nlm.nih.gov/nuccore/HM219844
PcChia1-1 http://www.ncbi.nlm.nih.gov/nuccore/HM219849
Also, we have 10ng of each Chitinase on the way from UBC.
The nucleotide and protein sequences for all chitinase clones are
available in NCBI GenBank. Accession numbers for all genes are listed in
the "Experimental" section of the paper under subheading "Nucleotide
sequence accession numbers". The expression vector that we used was pMal-c4x from NEB:https://www.neb.com/tools-and-resources/interactive-tools/dna-sequences-and-maps-tool . The genes were cloned into BamHI (right after) and before HindIII restriction sites.
Entry #25 Schematic Drawing of Project: Monday March 10
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Entry #26 DNA sequencing: Sunday March 23
Before Custom digest  | After Custom digest with our 5 restriction enzymes.  |
Entry #27: A quick link to our wiki and project proposal - Thursday, March 27
Entry #28: Paper by Dr. Raffa - Thursday, March 27
Entry #29: Workshop #2 - Saturday April 5
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Entry #30: Gene synthesis of Chitinase genes
Entry #31: Preparing Media and Growing Cultures & Why Won't My Cultures Grow?
Entry #33 - Wet Lab Experiment Thursday May 8 - Making competent cells and bacterial transformation
Entry #34 - Wet Lab Experiment Friday May 9 - Evidence and Analysis of Transformation of DH5-a Competent cells
 All chitinase plates had these "growths" on them; translucent, round-smooth "colonies", few in numbers, all grown on LB/Amp |  |  |  |
Entry #35 - Wet Lab Experiment Saturday May 10 - Evidence of Antibiotic Test; Transformation Redo
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a. concentrations aren't right
b. time in hot water bath not long enough - let's increase from 45 sec to 60 sec or let's decrease to 30 sec
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Entry #37 - Wet Lab Experiment Monday May 12
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Entry #38 - Process for making Chitin
Entry #40 - Sites with Lysis ideas
