Team:UFlorida/Experiments
Our Experiment
We first identified the genes comprising the pathway for tryptophol synthesis to be ARO9, ARO10, and ADH1 from the Saccharomyces genome database. These genes were then optimized for expression in E. coli and made compliant with iGEM RFC10 by the removal of illegal restriction sites. The genes were synthesized through IDT and ligated into DUET vectors. We chose DUET vectors because they have the T7 promoter for high protein expression. The plasmids with inserts were then transformed into DH5a E. coli cells for plasmid growth. Miniprep was performed on the DH5a cells in order to extract the plasmid, which was later transformed into BL21 E. coli cells for amplified protein expression. The BL21 cells underwent a cycled series of competency and transformation with a DUET plasmid containing a different insert. Overnight cultures from each transformation with another DUET plasmid were remade competent until we had all three genes of interest in one cell. We then tested for the presence of tryptophol in our cell culture using HPLC, and plated our cell culture against a fungus in the Chytridiomycota family.
For BL21 cells, only heat shock for 10 seconds
Chytrid Identification & Isolation
In order to combat a disease, one must first learn and study the disease to figure out its weaknesses. UFlorida got into contact with Dr. Matt Smith in the Department of Plant Pathology at the University of Florida. Dr. Smith has close relations with the UF Plant Disease Clinic and has experience working with chytrids. Since B. dendrobatidis is a virulent fungal pathogen, it was irrational to directly work with it in lab. Instead, another species from the same family, but far less threatening than Bd, would be sufficient to study. Following Dr, Smith’s advice we attempted to isolate and identify a chytrid species from the primary lake on campus.
Methods
1. Gather pond water from varying spots around the lake
2. Vacuum filtrate lake samples to remove organic matter (and mosquito eggs)
3. Pop popcorn kernels in microwave to expose less fibrous inner portion
4. Put kernels in water samples and let aerate for several days
5. Remove kernels into petri dishes
6. Rip off pieces and examine under microscopes
Materials
Media
Cellobiose - Tryptone agar (CT) 500mL
0.2g Tryptone
2g Cellobiose
5g Agar
DI water to 500mL
Antibiotics
200 mg/L Penicillin
300 mg/L Streptomycin sulfate
Microscopes
Dissection Microscope
Compound light microscope
MotiConnect app (picture capturing software for compound light microscopes)
Collection Log
September 5, 2017 - Chytrid collection
Baits created out of microwaved popcorn with 2 marbles in cheesecloth bags wrapped with hemp string. They were left in Lake Alice for 2 days.
September 20, 2017 - 1st Isolation Attempt
Figures 1 & 2. Fungal microbes obtained from Lake Alice
After an incubation period of 15 days, popcorn samples were placed into plastic petri dish to be examined under a dissection microscope. The samples were broken down to smaller pieces to make for easier examination and extraction. The spore with the discharge tubule sporangia and what appears to be zygotes were separated on to CT agar using a 20 microliter pipette. A marked the placement of the zygotes and B designated the site of the sporangia specimen. Upon further research, it was believed that both microbes from Figure 1 and Figure 2 were of the same species and they were at altering points of the diploid stage in its lifecycle.
September 27, 2017 - 2nd Isolation Attempt
Figure 3. Petri dish of isolated fungi from September 20th collection
Following a week for growth, both sites A & B grew colonies with red centers and white parameters. The colony at site A had a diameter of 0.4 cm and site B had a diameter of 1.5 cm. The petri dish was also filled with a white fuzz that covered the whole dish.
Figure 4. Close of red hyphae from site B
A sample of the red interior was taken to be further examined under a compound light microscope. Under closer examination the red color came from what appeared to be septate hyphae.
Figure 5. Mycelium from site A
The mycelium also had white hyphae protruding from the red hyphae. This could explain the white fuzz that filled the petri dish. A water mite is seen feeding on this fungi with distinguishable chelicera and a cephalothorax.
Figure 6. Close up of mycelium from Figure 5
Zoospores are seen being released in Figure 6. Three further isolations were conducted to identify if the white fuzz and the red colony were the same fungi only at different developmental stages. It was also necessary to question whether or not they were chytrids. Input was sought from Dr. Timothy James from the University of Michigan. He confirmed the presence and identified the morphological forms as oomycetes (water mold).
October 5, 2017 - 3rd Isolation Attempt
Figure 7. 2nd isolation of the unknown fungi
Upon isolation it was discovered that the ‘white fuzz’ was of a different species than the reddish colony in the center of the plate. When these species were separated they then grew without the reappearance of the other.
Figures 8 & 9. Oomycetes and microparasites.
Figures 8 & 9 show the water mold in the multicellular haploid stage of its life cycle. The green patches were noted by Dr. Matthew Smith to be microparasites feeding off the water mold colonies.Discussion
It was stated in the University Maine Chytrid Laboratory protocol (used for this experiment) that their method was more effective in the cooler months of the year. It was also noted that oomycetes over-run the baits when the water sampled is at warm temperatures. Because this experiment took place during the summer and in Florida, the result of obtaining oomycetes could have been predicted. Graduate students in Dr. Matthew Smith’s research lab suggested if the experiment were to be repeated, we could trying using pine pollen and snake skin as possible bait alternatives to increase the likelihood of catching a chytrid.
Sample Preparation
The standard stock solution (15 mg mL-1) of TOL was prepared in methanol. The standard Trp stock solution (15 mg mL-1) was prepared in water. Series (n = 5) of working solutions for each standard were prepared (0.0625 - 125 μg/ml)by appropriate dilution of the stock solution with methanol. To determine the recovery of the studied indolic compounds from bacterial broth during sample preparation, standards were spiked into sterile LB broth at five different concentrations. Bacterial broth was processed with the sample preparation procedure described below.1. The bacteria were cultivated for 72 h in LB broth with 0.5, 1.0, 2.0, 3.5 and 5.0 mM Trp supplementation
2. Bacterial cultures were then centrifuged, and the bacterial culture supernatants were processed
3. Sample preparation consisted of a single centrifugal filtration step using 3-kDa cut-off membrane centrifugal filters.
4. 0.5 mL of bacterial culture supernatants or spiked sterile bacterial broths were transferred to the sample chamber of a 0.5 mL centrifugal filter tube and centrifuged at 14,0009*g at 4 C for 30 min.
5. The filtrates were directly analyzed by HPLC.
HPLC Conditions
A C18 column was used after a previous run with a C8 column showed that tryptophan eluted too rapidly and early in the gradient method used.Eluent A
2.5 : 97.5 % (v/v) acetic acid: H2O, pH 3.8
*pH was adjusted by addition of 1 mol L-1 KOH
Eluent B
80 : 20 % (v/v) acetonitrile: H2O
Gradient Elution (A>B)
80:20
50:50
0:100
80:20
Time Scale(min) @ FR (1ml/min)
0-25
25-31
31-33
33-36
Analysis
Initial Standard Run of Tryptophan
MW of compounds:Tryptophan MM: 204.225 g/mol
Indole-3-pyruvic acid MM: 203.197 g/mol
Indole-3-acetaldehyde MM: 159.188 g/mol
Tryptophol MM: 161.20 g/mol
