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.
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
Tryptophan and Indole-3-pyruvic acid
Tryptophan and indole-3-pyruvic acid are too close in MM that unintended reactions during the run would make it difficult to differentiate the two.References
King ED, Ward MK, Raney DE, 1954. Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine 44, 301–7.