Team:CoBRA/Project

Restoring self-preservation in the lodgepole pine trees:

Inhibiting blue stain fungal proliferation using class I chitinase activity in transformed E. coli DH5alpha cells

   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. We optimized our DNA using BioBasics Inc.

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

  Competent Cells and Transformation
  After obtaining our optimized plasmids from BioBasics Inc , chemically competent cells were created using DH5α E. coli cells. Cells were grown in 5mL of LB broth at 37oC in a shaking incubator overnight. The following morning, 100μL of cells were grown in 9.9mL of LB broth and grown for 3-4 hours in a shaking incubator. Following the incubation cells were centrifuged and the supernatant was removed. The pelleted cells were then re-suspended in CaCl2 and cooled on ice for 30 minutes. Our plasmids were then added to the microcentrifuge tubes containing the DH5α cells and the tubes were placed in a water bath at 42oC for 60 seconds. This heat shock created pores in the cellular membrane thereby allowing the entry of plasmids. Our cells were then placed on ice for 5 minutes to close the pores in the membrane. Following this recovery, LB broth was added and the cells were placed in the shaking incubator for 60 minutes at 37 oC. After the incubation cells were then plated on agar plates and then incubated for 24 hours incubated. To determine whether our transformation was successful, antibiotics were added to our agar plates. The experimental plasmids contained antibiotic resistance and therefore only cells that had successfully been transformed with our experimental plasmids grew on the agar plates. These transformed cells were used to amplify our experimental plasmids.

Miniprep:
  Having amplified our plasmids, we next aimed to isolated them from our DH5α cells. In order to do this, a miniprep was performed using the E.Z.N.A. Plasmid DNA Mini Kit II (Omega Bio-tek Inc, Norcross, Georgia). DH5α cells were grown in LB and treated with RNase. Cells were then lysed, cellular debris was pelleted and the lysate was passed through a HiBind DNA mini column. Using the HiBind column, DNA was trapped as proteins while eluting proteins, membrane molecules and any other unwanted substrates. A wash solution containing ethanol is then spun through the column. The ethanol aided in precipitating the DNA. DNA was eluted using distilled water. The isolated plasmids were then visualized using gel electrophoresis. Plasmids were run on a 1.5% gel at 125V for 40 minutes. Molecular size of the plasmids were visualized using 1 kb Invitrogen 0.9% Ethidium bromide/Agarose and 1 kb Ready 1% TBE/Agarose DNA ladders.

Digest:
Having visualized that plasmid isolation was successful, we used the remaining product of our miniprep to perform a restriction digest of our experimental plasmids. The restriction digest was performed using restrictions enzymes SpeI and PstI for plasmids Pc1-1, Pge1-1 and Pge1-2, enzymes Xbal and PstI for plasmid J04450, and enzymes SpeI and PstI for plasmid J04500. To each chitinase minicentrifuge tube we added 30.5 uL of ddH2O, 5 uL of cutsmart buffer, 1 uL each of restriction enzymes and 12.5 uL each of Pc1-1, Pge1-1 and Pge 1-2. To each vector minicentrifuge tube we added 30.5 uL ddH2O, 5.0 uL cutsmart buffer, 1 uL each of restriction enzymes and 12.5 uL each of J04500 and J04450. We then gently pipetted each minicentrifuge tube followed by a centrifuge at 14,000 RPMs for 10 s. 20 uL from each tube was visualized using gel electrophoresis. Fragments were run on a 1.5% gel at 125 V for 40 minutes. Molecular size of the fragments were visualized using 1 kb Invitrogen 0.9% Ethidium bromide/Agarose and 1 kb Ready 1% TBE/Agarose DNA ladders. The remainder of our restriction digest was stored at -20oC for ligation.

Ligation:
  To deactivate our restriction enzymes, tubes were placed in a water bath at 80oC for 20 minutes. To ligase our parts into J04550 and J04500 we added 6.5 uL of each digested chitinase inserts (Pc1-, Pge1-1 and Pge1-2) to 2 uL of either the J04450 plasmid or the J04500 plasmid. We then added 1 uL of T4 DNA buffer and 0.5 uL of T4 DNA ligase. The ligation mixture was pipetted gently three times, spun down at maximum RPMs for 5 s and allowed to incubate at room temperature for 10 minutes. This gave us 6-0.6 mL centrifuge tubes containing our ligated plasmids.

  The ligated plasmids were then transformed into 100 uL of competent DH5a cells and left for 24 hours. We then grew up these ligated cells for miniprepping to obtain the six new constructs. From there the isolated plasmids were visualized using gel electrophoresis. Plasmids were run on a 1.5% gel at 50V for 120 minutes. Molecular size of the plasmids were visualized using 1 kb Invitrogen 0.9% Ethidium bromide/Agarose and 1 kb Ready 1% TBE/Agarose DNA ladders.

Proof of Concept:
  We will take some cells from a streak plate and incubate them in 15mL of LB broth and 5uL chlor for 12 hours. In a 1.5 mL centrifuge tube, we will put 1.2mL of culture and spin at 10000 rpm for 3 minutes, and then remove the supernatant. We will repeat this step until we have a large pellet. Once we have a large pellet we will resuspend in 750uL of LB broth. Next we will make 100mL of standard LB chlor plates (makes 2 plates) and add 100mg of practical grade chitin. This will serve as an assay for the production of chitinase, as these plates should have a cloudy color with chitin, but if the chitin is degraded they should be more transparent. We will place 7 drops of 60uL of our cells on these plates, and look to see if there is any color change

   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.

   At the time of submitting our wiki, we are in the process of proving proof of concept. We made very tiny agar plates containing 10 mg of practical grade chitin and grow a specified volume of our Pge and Pc cells on them. This will be a great introduction to exploring our question of whether our cells can produce chitinase, which will eventually lead to the use of chitinase in the degradation of the chitin-rich blue stain fungus cell wall.

Results

Figure 1. DIY Shaking Incubator: Hova-Bator Incubator Atop Fisher-Price Electric Plug-In Baby Swing

Figure 2. 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.

Figure 3. Successful Bacterial Transformation Plates Using DH5a E.coli May 12, 2014

Notice no strange fringe around colonies. Colonies are concise and dense.

Figure 4. Successful Ligation Transformation Plates Using DH5a E.coli June 3, 2014

Ligated DH5a with chitinase cells in J04500 backbone (pictured on left); Ligated DH5a with chitinase cells in J04450 backbone (pictured on right)

Figure 5. Gel Results of Digestion with Restriction Enzymes on J4450 Plasmids To Isolate Chitinase Protein in our DH5a E.coli cells with 1 Kb Invitrogen Ladder (June 14)

Moving from right the first well is a track it 1kb invitrogen DNA ladder, the second and third wells consist of a construct made up of PcChia 1-1 in a pSB1C3 backbone. In well 2 this construct was cut by restriction enzymes PstI and EcoRI, causing the chitinase coding region to become detached from the backbone. In well 3 this construct was only cut at the EcoRI cut site, causing the plasmid to be linearized. Wells 4 and 5 followed the same process as wells 2 and 3 with the exception that the chitinase coding region in wells 4 and 5 is of the PgeChia 1-2 variety. Wells 6 and 7 contain the PgeChia 1-1 gene. Using the ladder, our chitinase genes look to be approximately 1000 base pairs (lanes 2, 4, and lowest band), the pSB1C3 backbone appears to be approximately 2000 base pairs (lanes 2, 4, and 6 second lowest band), and the linearized construct looks to be about 3000 base pairs (lanes 3, 5, and 7). These numbers match up with our theoretical values.

2 uL ladder
4 uL loading dye + 20 uL plasmid digest
pH TAE buffer solution at 8 and at Room temperature

Figure 6. Gel Results of Digestion with Restriction Enzymes on J4500 Plasmids To Isolate Chitinase Protein in our DH5a E.coli cells from the Vector (June 17)

Moving left to right, lanes 1, 8 and 9 contain our two ladders, 1 kb Invitrogen 0.9% Ethidium bromide/Agarose (lane 8) and 1 kb Ready 1% TBE/Agarose (lane 1 6 uL of ladder; lane 9 12 uL of ladder). In well 2 this construct (PgeChia 1-1) was only cut at the EcoRI cut site, causing the plasmid to be linearized. In well 3 this construct (PgeChia 1-1) was cut by restriction enzymes PstI and EcoRI, causing the chitinase coding region to become detached from the backbone. Wells 4 and 5 followed the same process as wells 2 and 3 with the exception that the chitinase coding region in wells 4 and 5 is of the PgeChia 1-2 variety. Wells 6 and 7 contain the PcChia 1-1 gene. Using the ladder on the right, our chitinase genes look to be approximately 1000 base pairs (lanes 3, 5, and 7 lowest band), the pSB1C3 backbone appears to be approximately 2000 base pairs (lanes 3, 5, and 7 second lowest band), and the linearized construct looks to be about 3000 base pairs (lanes 2, 4, and 6). These numbers match up with our theoretical values.

2 uL ladder
4 uL loading dye + 20 uL plasmid digest
pH TAE buffer solution at 8 and Room Temperature

Discussion - interpretation

Our iGEM experiment purported that if we engineered an E. coli construct with a spruce/pine tree chitinase, then that transgenic E. coli will produce the tree chitinase. In doing so, our research will lend further support for a biological solution to a major ecological problem facing Canada and the USA.

Optimization of DNA

   To begin, our specific gene of interest, the chitinase, is not available in the iGEM registry. The cloned Chitinase genes were received from Dr. Bohlmann at UBC. The DNA needed to go through some additional changes before they were usable. 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. Use of NEBCutter website was beneficial in determining that our sequence was problematic and what was required to fix the situation. BioBasics Inc optimized our DNA so that the illegal cut sites were removed and we were able to utilize this DNA for our experimental purposes.

DNA Recombination Protocols

   Transformation of iGEM’s NEBTop 10 E.coli cells proved extremely difficult. In the end, after several attempts, it was decided that we switch to DH5-alpha E.coli cells. This strain has worked under our laboratory conditions. DH5 alpha cells maintain plasmids well and can be made competent on a more consistent basis. Modifications of the transformation protocol included suspending cells in CaCl2 on ice for 15 min prior to removing the second supernatant, increasing the spin RPMs in order to collect a greater volume of pellet while making cells competent, increasing the heat shock 45 s, floating on ice for 5 min, increasing the volume of solution taken for growth on plates, addition of more luria broth media before growing colonies on plates and including control plates along with the experimental plates. Modifications to our miniprepping protocol included increasing our volumes of ingredients added to the culture tubes and ensuring that the dry spinning was achieved before adding the elution buffer. Restriction digest and ligation protocols were following specifically using addgene website and iGEM methods. For identification of plasmids or genes we used gel electrophoresis. We had issues with the integrity of our bands and ladders and consequently with our purported results. Part of the issue was that our plasmids floated out of the wells while loading which meant that we did not dry spin the centrifuge column enough. We corrected this step and had greater success as seen in Figures 5 and 6. As for the choice of ladders, it appears from Figures 5 and 6 that the 1 kb Invitrogen 0.9% Ethidium bromide/Agarose DNA ladders worked the best in our TAE buffer solution and in our high school laboratory conditions.

Gel Results

   Looking at our data, we believe that the length of the parts that were restriction digested with two enzymes should add together to equal the length of the parts that were restricted with one enzyme. By the ladder on the right of both Figures 5 and 6, our chitinase genes look to be approximately 1058 base pairs for PgeChia 1-1 and PcChia 1-1 and 1067 base pairs for PgeChia 1-2 (1), the pSB1C3 backbone appears to be approximately 2070 base pairs (2), and together the constructs look to be about 3128 base pairs for PgeChia 1-1 and PcChia 1-1 and 3137 for PgeChia 1-2. These lines match up with our theoretical values. The genes for the production of chitinase were the same ones tested in Kolosova, N., et al (3).

Proof of Concept

   We are currently in the process of attempting to prove the production of chitinase from our transformed DH5alpha cells in the presence of lab grade chitin agar plates. Results to be determined.

Limitations

   Accessibility to high tech equipment and critical chemicals was a hinderance with the wet lab component of our project. For example, we do not have a optical density machine, which made many of our calculations that required knowledge of how much plasmid our solutions contained unpredictable. Also, as we try to achieve proof of concept, we are missing some of the compounds that would help us make a different kind of agar plate that is optimal for testing for the breakdown of chitin. Furthermore, looking at our gels that we ran in the results section, although the lines look to be close to where they would be expected to be, they are not exact. We see two lines where the double digested parts are and one line where the single digested parts are, but these lines are not exactly where they should be in regards to the ladder. Seeing the rounded edges of the lines in our gel, we believe that our TAE buffer may be have been incorrectly made, which could cause our samples to run incorrectly.

Significance of Corroboration

   Despite our relative inexperience in the field of genetics and phytochemistry we were presented with the opportunity to work alongside Dr. Bohlmann and Dr. Kolosova, two researchers at UBC’s Michael Smith Laboratory and members of the TRIA project. Their coauthored paper on chitinase and their in depth knowledge of phytochemistry, the mountain pine beetle and the blue stain fungus proved to be a very valuable resource to our team as we set out to test chitinase production in transgenic E.coli4.

   Our project, although parallel to the work done by Dr. Bohlmann and Dr. Kolosova, has provided iGEM with 6 new chitinase biobricks that will go on to aid future teams in their endeavors. In addition to our lab work we have also completed a high school level lab manual which combines tested lab protocols with high school level terms and directions, thus making synthetic biology more accessible to high school students. In the context of iGEM we have blazed a trail by creating six new BioBricks in a single iGEM cycle and providing iGEM with a multitude of high school level protocols to make synthetic biology more applicable and accessible at the secondary level

Areas for Future Studies

   Our project is by no means ready to be put into a real world application. Firstly, the cell walls of the blue-stain fungus are not made exclusively of chitin. In Kolosova et al3 Phytochemistry, when the chitinase was tested on the blue-stain fungus, no antifungal activity was observed, as the cell walls contain many more compounds which are not degraded by chitinase. In future years, we may research these compounds, and see if we can alter our plasmid to also degrade them. Also, we currently are using the LacI promoter in our constructs. This promoter will not function if IPTG is present. For the forest ecosystem, this could be hazardous, as there are many beneficial fungi in forests which we would risk killing. For next year we hope to try and find a promoter which functions only when the tree is producing stress hormones such as terpenes or other indicators. This would help the tree fight off the infections with minimal, if any damage to the other fungi of the forest. Additionally, we need to find a method of delivery of our plasmid. The DH5alpha cells that we used are not able to survive in an environment such as a forest. Hopefully we could use a bacteria that is naturally present in the tree, to avoid introducing an alien species to the forest. Producing chitinase in E Coli is just the first step in combating the blue-stain fungus.

Work Cited

Vancouver Referencing Guides
1. http://store.biobasic.com/
2. http://parts.igem.org/Part:pSB1C3
3. Kolosova, N., et al. Cloning and characterization of chitinases from interior spruce and lodgepole pine. Phytochemistry (2014).
4. http://www.for.gov.bc.ca/hfp/mountain_pine_beetle/