Section 1: Purpose & Flow of Experiments

Here we outline the general flow of our experiments and their purposes for testing our system.

There were two major lab experiments: successful transformation of designed parts into bacteria, and testing of the final components and system.

Transformation of engineered bacteria

The majority of our time spent in the lab this summer was related to transforming our bacteria with our designed parts and receptors. We began by individually working on each metabolite receptor system, ensuring that the receptors were functional and would respond to the metabolites accordingly. The following list are example procedures that were carried out to begin this process:

  1. Amplifying sequences via PCR
  2. Gel electrophoresis
  3. Gel Extraction
  4. RE digest
  5. Ligation
  6. Miniprep
  7. DNA transformation
  8. Growing bacterial colonies

Unfortunately, we were unable to complete these systems prior to Jamboree, as we faced difficulties with ordering materials which hindered our timeline. However, we did manage to successfully amplify sequences via PCR, run Gels, Extract DNA from Gels, Grow bacterial colonies, and Miniprep Plasmids from bacterial cells, all of which will be elaborated on in the next section.

Testing engineered system

As we were still working on validating our transformations, we did not reach the second part of the experiment that allows us to test the system as a whole. We plan on completing these experiments in the following year.
The following is a list of planned experiments:

  • Basal expression readings
    • No indole, GABA, or butyrate in the cell and measuring basal GFP, BFP, and RFP
  • Input to output time
    • Establish timing between input/addition of metabolites and output fluorescent readings (note half-life decay time of fluorescence if applicable)
  • Fluorescent decay timing
    • How long does it take for the FPs to decay? How does this correspond to the input to output time?
  • Testing fragments individually and together
    • Testing indole fragment through measuring GFP expression
    • Testing GABA fragment through measuring BFP expression
    • Testing butyrate fragment through measuring YFP expression
    • Testing LuxI/R (only indole) through measuring GFP and RFP expression
    • Testing high indole, low GABA, we expect to see high GFP, low BFP, high RFP expression (also testing coactivation by HrpS/HrpR in CELL1+2)
    • Testing low indole, high GABA, we expect to see low GFP, high BFP, no RFP expression (also testing HrpV inhibition in CELL1)
    • Testing high indole, high GABA, we expect to see high GFP, high BFP, no RFP expression
    • Testing high indole, low butyrate, we expect to see high GFP, low YFP, high RFP expression (also testing HrpV inhibition in CELL2)
    • Testing low indole, high butyrate, we expect to see low GFP, high YFP, no RFP expression
    • Testing high indole, high butyrate, we expect to see high GFP, high YFP, no RFP expression
    • Testing high GABA, high butyrate, we expect to see high BFP, high YFP, no RFP expression
    • Testing butyrate sensitivity by adding other SFCAs
  • Dose response curves
    • Percent-fold increase in GFP after 25%, 50%, 75%, and 100% fold increase in [indole]
    • Percent-fold increase in BFP after 25%, 50%, 75%, and 100% fold increase in [GABA]
    • Percent-fold increase in RFP after 25%, 50%, 75%, and 100% fold increase in [butyrate]
    • Establish saturation effects
      • Indole, GABA, and buyrate minimum and maximum threshold concentrations
    • LuxI to 3OC6HSL turnover rate via activation of LuxR and subsequent coactivation of PHrpL and CFP

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