Results

Experiments Conducted and Their Methodology

Experiments

Insertion of DNA Into E. Coli Bacteria to Cause Bioluminescence

Objectives of the Experiments

Our experiments had the main objective of inserting DNA into E. coli bacteria; this bacteria with the inserted DNA was mixed into the soil causing an emission of bioluminescence in case of the presence of AHL in the soil. This bioluminescence is what would be detected by our hardware to signal the presence of Fusarium oxysporum.

Specifically, we used genetically engineered plasmid DNA to be placed inside of the bacteria’s genetic code.

We started by extracting the plasmids with a MiniPrep procedure and creating copies of the plasmid DNA through PCR.

E. Coli

Escherichia coli (E. coli) is abundantly found in the soil of subtropical and tropical environments - bananas grow in these kinds of environments.

E. coli is also a model organism, meaning that there are many available studies available for the bacteria. This makes E. coli an ideal candidate for our experiments. The strain of E. coli that we used, DH5a, was particularly useful to us because of the ample research conducted on it. The strain is proven to have a high transformation efficiency and yield of DNA, this gene is likely to be the most productive.

But how did we introduce DNA into E. coli?

This was done through electroporation of the E. coli cell membrane and the insertion of plasmid DNA into the temporary pore.

We confirmed the presence and stability of the plasmid DNA inside of the bacteria through 3 steps.

  1. Agarose gel electrophoresis - we placed amplicons (small pieces of DNA) inside of an electrophoresis device.

    2. The device consists of a gel that has an electric current flowing through it; smaller amplicons move through the gel faster and end up further right while the larger amplicons move through the gel more slowly and end up on the left side of the device. We compared what the supposed location of the amplicons are to where our amplicons were really located on the electrophoresis device to decipher whether the sequence was accurate. We repeated this process with the new plasmid copy to ensure that there are no post-transcriptional mutations in the original sequence.

  2. We performed cell lysis and the Bradford Method - after incubation, we broke down the bacteria’s cell membrane and used polyacrylamide gel electrophoresis to separate the proteins. We identified the plasmid DNA through the Bradford Method.
  3. We put the E. coli in contact with chitin, which is confirmed to induce the expression of bioluminescence in the bacteria.

Gene Expression Assay

Main objective: to find the most effective E. coli strain as well as the most effective genetic circuit for plasmid DNA insertion.

For the validation, standardization, and development of the mathematical model associated with the gene expression system, we aim to identify and standardize the expression conditions by using the DH5a E. coli strain and a genetic circuit, both already employed and validated in synthetic biology assays.

Preparation

  1. Obtain the DH5a bacterial strains.
  2. Prepare one genetic circuit with 1 variation and one genetic circuit with 2 variations.
  3. Prepare Petri dishes with Luria-Betrani (LB) Broth.

Procedure

  1. Inoculate each strain with a respective genetic circuit in LB medium.
  2. Incubate 72 hours under appropriate conditions: in the dark at 21℃.
  3. Conduct a test for optical density using a spectrophotometer.
  4. Record gene expression levels for each replicate and condition.
  5. Conduct test for fluorescence level through electrophoresis.
  6. Record gene expression levels for each replicate and condition.

Genetic Circuit Testing

Test for Genetic Circuit #1 (tested with 1 variation)

  • The DH5a bacterial strain will undergo 12 tests

Test for Genetic Circuit #2 (tested with 2 variations)

  1. Variation 1: containing a non-mutated LuxB genetic sequence
  2. Variation 2: LuxB sequence combined with Venus Mutation; meant to amplify the bioluminescence of the E. coli when in contact with AHL
  • The DH5a bacterial strain will undergo 12 tests for each variation of the genetic circuit for a total of 24 tests.

Negative Controls

  • To understand how the bacteria behaves under natural circumstances, the E. coli will grow inside of an LB broth without the genetically engineered plasmid.
  • Demonstrating how the medium behaves under natural circumstances without the deliberate growing of bacteria, one sample will be the LB broth with no bacteria on it.

The total number of tests, including those for both genetic circuits and the negative controls will be 38.

Analysis of Variance

A statistical analysis software, called ANOVA, will analyze the statistically significant differences among the various treatments.

Growth Kinetics Assay

The main objective of the assay is to stimulate the pathogenic effects of Fusarium Oxysporum through the contact of chitin in order to obtain the kinetics rate of the E. coli.

The Growth Kinetics Assay will help validate the mathematical model created by our math team.

The 1st circuit and the second circuit were analyzed as a whole unit.

The 2nd genetic circuit was tested with two variations of the LuxB

  1. Variation 1: LuxB
  2. Variation 2: LuxB combined with Venus Mutation; meant to amplify the bioluminescence of the E. coli when in contact with AHL

Important Notes

  • Experiments will be performed in vitro as the pathogens may pose health risk to lab workers.
  • Plants produce antibodies against pathogens like Fusarium oxysporum. Chitin is one component of the cell walls in F. fusarium. A study tested a synthetic antibody against chitin (as the presence of chitin would mean the presence of the pathogen). The synthetic antibody binds to the chitin, which allows researchers to determine the amount of chitin. The amount of chitin used in the study should be proportional to the amount of fusaric acid produced by F. fusarium.

Preparation

Material for Preparation

  • 3 different concentrations of chitin: 10 mg/ml 1 mg/ml 0.1 mg/ml
  • Plasmid DNA encapsulated in lipofectamine
  • 100 μL of culture with 1x 10^7 of DH5a E. coli strain

Preparation of Materials for Kinetics Test

  1. Samples (including the chitin and E. coli culture) incubated in the dark at 21℃ for 72 hours
  2. E. coliwashed in 1% Tryptone
  3. Fluorescence tests conducted on samples

Preparation of Samples

  1. Test of the 3 concentrations of chitin on DH5a E. Coli strain #1 (DH5a)
  2. Repeat the test 3 more times for a total of 4 trials.
  3. Cell growth and fluorescence measured for each trial, every hour for up to 16 hours after the end of the trial.

Controls

  • 3 bioreactor tubes with the LB Broth and strain.
  • All circuit combinations.
  • LB Broth with no bacteria

Procedure

  1. Each strain inoculated in LB medium and LB medium with genetic circuits.
  2. Growth monitored by measuring optical density (OD) at regular intervals using a spectrophotometer.
  3. OD values recorded for each replicate and condition.
  4. Bacteria placed inside of bioreactors with different concentrations
  5. Samples placed in a bioreactor to measure OD.
  6. Changes in fluorescence observed over time.

Each test has 12 replicates.