The goal of this assay is to determine if a 'predator' bacterial strain is effectively secreting a microcin to kill a 'prey' bacterial strain. The assay includes a control strain which does not secrete microcin, the predator strain which secretes the microcin, and the prey strain which is susceptible to the microcin. A zone of inhibition is a clearing of the prey strain around a predator strain spot which is caused by the predator secreting microcins that kill off nearby prey. If a plated bacteria shows a zone of inhibition, it is secreting the microcin.
Day 1:
In sterile culture tubes, prepare overnight liquid cultures of the following strains:
Day 2:
Media and Bacteria Volume Calculator:
Strain
Final OD600
Final Volume (mL)
Measured OD600
Volume to Add (mL)
Prey
0.1
5
Prey OD600
0.1 * 5 / Prey OD600
Predator
50
0.1
Predator OD600
50 * 0.1 / Predator OD600
Our experiments focused on the center rot of onions that affected the Texas 1050 sweet onion. Researchers had found that specific strains of Pantoea caused center rot while different Pantoea strains had been used as biocontrols. Our goal was to engineer the symbiont strains of Pantoea to secrete microcins that would target the pathogenic Pantoea strains. The specific strains we were working with are listed below:
Strain Name
Relation to onion
Pantoea vagans C9-1
Biocontrol
Biocontrol
Pantoea agglomerans PNG 92-11
Pathogen
Pantoea allii PNA 200-10
Pathogen
Pantoea ananatis LMG 2665 (ATCC 33244)
Pathogen
Pantoea ananatis PNA 97-1R (Rif resistant mutant)
Pathogen
These specific strains were acquired through the collaboration and generosity of the Davies lab at the University of Texas at Austin.
To assess whether our engineered symbiont would effectively treat the center rot in onions, we needed a protocol that would allow us to induce center rot in onions and track its progression over a period of time.
Initially, our protocol was adapted from Dr. Schroeder, a plant pathologist at the University of Idaho, but we struggled with growing a sufficient amount of pathogenic Pantoea strains in the Texas 1050 onions. Using the knowledge we gained from our conversations with Dr. Brenda Schroeder, we were able to adapt her protocol for inducing center rot in onions.
Day 1:
Day 2:
Day 3:
Day 1:
Program should be as follows:
Step
Temperature
Time
1
42°C
1.5 min
2
16°C
3 min
Cycles 1-2
-
Repeat 30x
3
60°C
5 min
If you are planning to run the reaction overnight, use this program instead:
Step
Temperature
Time
1
42°C
5 min
2
16°C
5 min
Cycles 1-2
-
Repeat 30x
3
60°C
5 min
Day 2:
Day 3:
Day 4:
Day 1: Setting Up GGA Reaction(s)
DNA Component Ratios for Each Assembly Reaction
DNA Component
Desired Moles (fmol)
Plasmid Length (bp)
pBTK1028
25
3446
Type 2 Part Plasmid
50
-
Type 3 Part Plasmid
50
-
Type 4 Part Plasmid
50
-
Reagent Volumes for Each Assembly Reaction
Reagent
Volume
dH2O
-
10X T4 DNA Ligase Buffer
2
pBTK1028/backbone
-
Type 2 Part Plasmid
-
Type 3 Part Plasmid
-
Type 4 Part Plasmid
-
NEB Golden Gate Assembly Mix (BsaI-Hfv2 and T4 DNA Ligase)
1
Total
3
Program should be as follows:
Step
Temperature
Time
1
42°C
1.5 min
2
16°C
3 min
Cycles 1-2
-
Repeat 30x
3
60°C
5 min
If you are planning to run the reaction overnight, use this program instead:
Step
Temperature
Time
1
42°C
5 min
2
16°C
5 min
Cycles 1-2
-
Repeat 30x
3
60°C
5 min
Day 2: Transforming into Component Cells
Day 3: Checking on Transformants and Screening Plasmids
Day 4: Examining Cultures, Making Stocks, and Plasmid Miniprep