CoBRA wiki

Entry #1: Below is a link to our project guidebook. You can jump to the specific section for your job.

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?

Entry #3: Correspondence Between Adam Sibbald and Dr. Matt Bryman (January 19th 2014):

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:

- AESRD MPB website:

- CFS website:

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.



Matt Bryman
Network Manager

The NSERC TRIA Network (TRIA-Net)

Entry #4: Correspondance Between Ms Bennett and Dr Janice Cook, TRIA Project Lead, University of Alberta

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,

Associate Professor
Department of Biological Sciences
University of Alberta

Entry #5: Mountain Pine Beetle

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.

Entry #6: Genome Of Blue-Stain Fungus Evolved To Bypass Tree Defence In Mountain Pine Beetle Epidemic

"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."

Entry #7: Pheromone Production In Bark Beetles

"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
This site explains how verbenone can be put to use to combat the mountain pine beetle. This chemical is a temporary solution and only works in places where the beetle has not completely infested the area.

Entry #9: Beetle facts
Interesting facts about the beetle dealing with winter temperatures at the bottom of this page.

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

“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

  • 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.

  • 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.

Great website from Princeton about Chitinase. Definitely worth a read.

Entry #13: Female pine beetle pheromones

Found an article relating to Entry 11. It states that the aggression pheromone is called Trans-Verbenol

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

Entry #16 Chitinase experiment
In this experiment, a chitinase is expressed by an e coli bacteria

Entry #17 The Economic, Social and Environmental Costs of the Mountain Pine Beetle
Stage One Report_The BC Experience and Lessons for GAER (Final)_ January 2007.pdf

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 ( )

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

There seems to be an overall concern regrading the sustainability of Dr Cooke's suggestion. How to keep that suicidal gene going in the BSF. So to build on that, here's Dr Bohlmann's suggestion and Magda's response to that.

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: (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.
Assuming that (1) the team can use Agrobacterium to introduce a chitinase gene into the BSF, and (2) the engineered BSF would keep the gene, my question is: How do you get the transgenic BSF onto the trees? Would the plan be to add the transgenic fungus to already infested trees, and hope that it would not only destroy itself but the wild type BSF too? Or would the plan be to replace the wild type fungus with the transgenic one altogether (would that even be doable at all)? Unless I'm totally off track here, I am not sure how feasible or safe any of this can be made.
Dr. Bohlmann's message seems more interesting to me. I couldn't open the link he sent (gives me an error) but from what I understand his lab is about to publish data on expression of spruce and pine tree chitinases in E coli (under IPTG induced promoter). He doesn't say if it worked or not or if it did, how well, but it seems to me that is the avenue worth pursuing.
Maybe you can pick his brain a little more re the paper he is about to publish? Maybe his lab managed to get some expression and you can ask him to send you the gene(s) so you can use a different promoter? I am going to go back to what I said in an earlier message: simple is better. My suggestion would be to start with a working (maybe even constitutive) promoter and a chitinase that worked in a previous (dr Bohlmann's?) E coli construct. You could also compare with different chitinases from the registry? Add a secretory tag (Lisa's earlier point)? I think there would be plenty of interesting stuff to try in the relatively harmless laboratory E coli before attempting to engineer the fungus that is known to harm the trees.

Source: Wikipedia
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
From Magda
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).
From what I understand, you'll get quite a few constitutive promoters in your kit, either by themselves or with additional regulatory sequences for fine tuning of expression. For example, this is a promoter part by itself:, while this is a composite part for high expression: You will also get this promoter:, which behaves as a constitutive promoter unless you engineer the cells to also express its repressor (TetR).
Therefore, even without trying to replicate dr Bohlmann's results, my feeling is that the team would have plenty of interesting things to try once you get your hands on a working chitinase construct.

A secretory tag would be a protein fragment that would be attached to your protein of interest (the chitinase), and would serve as a signal for the cell to export/secrete that protein. Essentially, if you need your protein to be secreted, you fuse its coding region with the coding region for the secretory protein. For your purposes, it would be important for the chitinase enzyme to get outside the bacteria, which is where its substrate/target would be (ie the chitin in the cell wall of the fungus). The alternative would be to build in a suicide-switch along with the chitinase, so cells will produce the chitinase and then would lyse to release/expose the enzyme to allow it to access the chitin.
I searched the registry but was unable to find a secretory tag - it's probably because of my limited knowledge of the registry and how it works. One part I found that looks of interest to me is this:; It's in your distribution kit but it's not for secretion. It is for display of the protein of interest on the outer cell surface. I don't think it's ideal (to have the enzyme displayed may constrain/impair its activity) but that's all I could find. I am sure that experienced iGEM-ers will have a lot more ideas on this.

Entry #21 Meeting with David and Himika - February 25, 2014

Entry #22 Origin of the chitinases - Wednesday March 8
The chitinases were cloned from interior spruce (Picea glauca x engelmannii) and lodgepole pine (Pinus contorta) trees.
This information was found in the second sentence of the abstract of Bohlmann's most recent paper. ( "cDNA for six chitinases were cloned from interior spruce (Picea glauca x engelmannii) and four from lodgepole pine (Pinus contorta)." ) The next sentence contains the names of the six chitinases from the interior spruce and the four from the lodgepole pine. Of the three chitinases that we are looking to get from Dr. Bohlmann, two were cloned from the interior spruce (PgeChia1-1 & PgeChia1-2) and the last was cloned from the lodgepole pine (PcChia1-1).

Entry #23 Ken Raffa
This is a link leading to the website of Ken Raffa, a leading Entomologist who has an advanced understanding of the forest ecosystem. His expertise was recommended to us by Dr. Bohlmann

Entry #24 Nucleotide/ Protein Sequences: Saturday March 8




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: . The genes were cloned into BamHI (right after) and before HindIII restriction sites.

Entry #25 Schematic Drawing of Project: Monday March 10

Entry #26 DNA sequencing: Sunday March 23
1. Template from sent to us by Lisa O.
2. Open, NCBI website where gene sequence is located, Google Help:Biobrick Prefix and Suffix from iGEM, NEBCutter V2.0

Checking on Cutsites in your DNA:
a. NEBcutter have open
b. Open Help:BioBrick Prefix and Suffix in iGEM - copy and paste prefix (gaattcgcggccgcttctag - first tag) into NEB text box.
c. Open NCBI - click on FASTA - copy and paste chitinase class gene into NEB text box after prefix
d. Open Help:BioBrick Prefix and Suffix in iGEM - copy and paste suffix (tactagtagcggccgctgcag- only tag) into NEB text box after chitinase gene sequence
e. Hit "submit" (right hand side) for cutsites in NEBcutter
f. Go to bottom right - click "custom digest"
g. Click on restriction enzymes: XbaI, SpeI, PstI, NotI, and EcoRI
h. Click on "digest"
You will now see only those cut sites and can see where the restriction enzymes cut into your gene. We will also get the exact number of nitrogen bases in our gene sequence.

Before Custom digest
After Custom digest with our 5 restriction enzymes.

3. Fill in Gene sequencing sheet as per Lisa's instructions. Used PgeChia1-2 as template.
4. Fill in Gene synthesis optimization request as per Lisa's instructions. This is for the protein sequence. Use NCBI - choose protein id= click on its link ; choose FASTA; copy and paste into optimization sheet.

Entry #27: A quick link to our wiki and project proposal - Thursday, March 27

Entry #28: Paper by Dr. Raffa - Thursday, March 27

This paper by Dr. Raffa explores the relationship between the fungi, beetles, and trees.
The paper suggests that there are many factors that determine whether a tree is able to fight off the beetle / fungus combination such as "vigor of the tree, site and stand conditions, and the size of the beetle population".
So despite the fact that some conifer species have naturally occurring chitinases, these tress could still die in a high density beetle infestation by the fungi contributing to the death of the host tree through mycelial penetration of host tissue or the combined action of toxin release and its effect on conifer defenses. Therefore, our project may not be strong enough to aid the trees in areas of high beetle population.
On the other hand, the paper seems to suggest that a healthy population of trees would be able to resist the fungi and beetles if the beetle population is low enough.
The paper also confirms that our prediction that "beetle-caused death of any particular tree is relatively low"

This experiment with a compound called chitosan was used to increase resin output from the tree. It shows that sheer resin output will overwhelm and force out any fungi carrying beetles. As a result, the pine beetle egg count in the tree was reduced, and blue stain fungus was not found the the trees that were treated with this chitosan. It is unclear whether this experiment was performed in a high density or low density beetle population. It is also unclear whether this experiment had any effect on the surrounding forest community, like woodpeckers.

Entry #29: Workshop #2 - Saturday April 5
Still need to do the following:
1. Shaking incubator - DIY
2. Timers for Day 3
3. NEB Biobrick enzymes - E, X, S, P, buffer and ligase
4. BioBasics - chitinase enzymes

Entry #30: Gene synthesis of Chitinase genes

The Chitinase genes that we received from Dr. Bohlmann 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. Here are some screenshots of the original parts from the NEB cutter site, with all PstI, EcoRI, XbaI, SpeI and NotI cut sites displayed.

PcChia 1-1

PgeChia 1-1

PgeChia 1-2

As you can see these parts have illegal cut sites in them. We sent the base pair sequence and protein sequence to Biobasics and asked them to invoke a silent mutation where the illegal cut sites are, and to add the biobrick prefix and suffix. Here are the resulting cut sites on the genes.

PgeChia 1-1 and PcChia 1-1 (Identical pictures)

PgeChia 1-2

The illegal cut sites have been removed and the biobrick prefix and suffix have been added.

Entry #31: Preparing Media and Growing Cultures & Why Won't My Cultures Grow?

Preparing media and growing cultures :

Why won't my cultures grow :
Entry #32 - Wet Lab Experiment Wednesday May 7 - Inoculation
1. Inoculated 5 mL LB media (broth) with a single colony of DH5a cells from petri plate into a 15 mL Falcon tube.
2. Incubated each falcon tube in shaking incubator at 37 deg celsius overnight.

Entry #33 - Wet Lab Experiment Thursday May 8 - Making competent cells and bacterial transformation
1. 8 am added 100 uL of DH5a mixture to 9.9 mL LB liquid media in to a new 15 mL falcon tube. Did this for two tubes.
2. Placed new falcon tubes into shaking incubator and will leave for 6 hours at 37 deg celsius in shaking incubator.
3. 4 pm made Competent cells (Chitinases - added 40 uL ddH2O to each vial diluted to 1:10 then used 2 uL; RFP - added 500 uL ddH2O to 50 pg vial - all added; J04500 - added 10 uL to spot on kit - all added)
4. 5:30 pm began transformation - made a note that we were working with a very, very small pellets
5. 7 pm incubated 5 plates overnight

Entry #34 - Wet Lab Experiment Friday May 9 - Evidence and Analysis of Transformation of DH5-a Competent cells
Evidence for Test Trial #2

All chitinase plates had these "growths" on them; translucent, round-smooth "colonies", few in numbers, all grown on LB/Amp
Colonies should look like this
All iGEM DNA (J04500 and J04450) colonies looked like this; strange peripheral edges; more transparent on edges not uniform solid growth; J04450 did not flouresce red as can see by negative colour under purple light
RFP colonies should be red within 18 hours at 37 deg C

1. All 5 plates did not transform properly. Confirmed by Lisa O, David L and Dr Bohlmann (emailed this evening)

1. Grow up another vial of DH5-alpha cells for another transformation
2. Check for negative growth on LB/Chlor media
3. Double check concentrations - used 1:10 diluted Pge1-1, Pge1-2, and Pc1-1; used 100 ng/uL of RFP; used less than 100 ng/uL of J04500

Entry #35 - Wet Lab Experiment Saturday May 10 - Evidence of Antibiotic Test; Transformation Redo
1. 7 am added 100 uL of DH5a mixture to 9.9 mL LB liquid media of each to three falcon tubes. Incubated in shaking incubator.

Evidence of Antibiotic Test

1. Tubes on far left show negative growth in each. DH5a cells from LB plate were inoculated in LB/Chlor showing no growth as the antibiotic killed them. The second tube is LB/Chlor with no cells and showing no growth. The antibiotics are sterile.
2. Tubes in middle picture show positive growth of our DH5a cells in LB media.
3. Tubes in far right together illustrating cloudiness we're looking for.

Conclusions for antibiotic test
1. Growth is not being compromised by our Chloramphenicol antibiotic
2. Transformation failure could be because:
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
c. need greater pellet size when making competent cells.

Transformation Lab
1. Performed a control (LB) and test (LB/Chlor) for both competent and transformation lab.
2. Tried two different concentrations for the Chi-DNA; 10 ng/uL and then diluted it 1000X to 1pg/uL
3. Heat shocked for 60s and on ice for longer
4. Changed up the competent test by putting on ice before adding last 100 uL of CaCl2

Results from Saturday's Test Trial #3

Entry #36 - Wet Lab Experiment Sunday May 11 -

Results from Sunday's Test Trial #4

1. DH5a cells have been compromised
2. Antibiotic plates don't seem to be working correctly

1. Make new sleeves of antibiotic plates, in particular Amp and Chlor
2. Use newly arrived competent DH5a cells and transform them with our chitinase plasmids.

Entry #37 - Wet Lab Experiment Monday May 12

Results After Growing New Antibiotic Plates and Receiving New Competent DH5a cells

1. All 6 control LB and experimental LB/Antibiotic plates saw superb colony growth
2. All LB/antibiotic plates with no cells had no growth showing us our antibiotic plates are working
3. Both LB plates with no cells had no growth showing us our LB media is not contaminated.

1. Inoculated LB media with DH5a cells from the LB plates - this is to grow a stock of DH5a cells for competency
2. Restreaked 6 new antibiotic AMP plates to get better colony growth in preparation for Miniprep tomorrow

Entry #38 - Process for making Chitin

This is a process for making chitin. We could use this chitin to achieve proof of concept

Entry #39 - Chitin for Sigma Aldrich

This is a link to a site at which we could buy chitin from shrimp shells

Entry #40 - Sites with Lysis ideas

These sites have ideas for a way to lyse cell while leaving the protein intact

Future goals

Idea #1
  • 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)

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.
An alternative would be something like this 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 ( 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

Idea #3 - Saturday March 29 from Richard Lee

Arming Trees Against Pine Beetle Invasions