Team:UGA-Georgia/Notebook
Notebook
MAY
Week 1
Made plasmids and prepared to test our constructs
Made LB broth and ampicillin agar plates
Prepared for transformation, plating, and extraction
Week 2
Determine if the insert is present within the transformed expressor plasmid
Did not see an insert
Week 3
Determine if we need to take any steps to visualize the GFP: UV light?
Prepared bacterial stocks of parent plasmids transformation completed
Begin examining the literature for aflatoxin detection strategies using the concentration of aflatoxin most prevalent in nature
Week 4
Digestions, gels, and gel purifications
Ligations (for both repressor and expressor)
Ligated plasmids transformed into DH3α cells
Minipreps of parent plasmids from iGEM were performed protocol is in the miniprep box.
JUNE
Week 1
Digestions, gels, gel purifications, and ligations
Ligated plasmids were transformed into DH3α cells
Minipreps of parent plasmids from iGEM were performed via protocol in the miniprep box
Week 2
Gel Purification of 4 Vector digestion: each vector digested with EcoR1 and Pst1 +/- CIP
Column Purification of Insert digestions EcoR1 and Pst1
Ligations were performed and cells were transformed with litigation mixture
No GFP expression seen from our expressor
Double transformations done using expressor and repressor obtained from minipreps; double antibiotic (A & C) plates were used for selection.
Week 3
Updated sequences for expressor, repressor, expressor 3’ Truncated by 9 bp, Expressor 5’ Truncated by 9 bp, and expressor with aptamer.
Week 4
Picked and inoculated fluorescent colonies from the E31 streak plate. Picked and inoculated some fluorescent and non-fluorescent colonies from the E3 and R spread plate.
JULY
Week 1
Extracted plasmids from previous week’s samples, digested appropriately, and visualized on a gel.
Week 2
Met with PI about protocols for working with aflatoxin
Week 3
Digestion of double E3/Repressor construct to show presence of GFP/Expressor in gel
Week 4
Interlab: Scheduled date to use plate reader to measure fluorescence.
Made 16 LB Agar plates with chloramphenicol
AUGUST
Week 1
Interlab: Made fluorescent stock; followed the Interlab protocol to prepare and plate samples and controls to be incubated.
Week 2
Interlab: all plates except for the negative (as expected), TD 2, and TD 3 grew colonies. Made 16 more LB agar plates with chloramphenicol for replating of TD 2 and TD 3. Replated TD 2 and TD 3; no growth on either plate
Week 3
Interlab: resuspended DNA from Kit 7 for TD 2 and TD 3. Replated TD 2 and TD 3 with new DNA; transformations were successful.
Autoclaved LB broth in preparation for picking colonies
Counted colonies for control transformation: 4 colonies grew on the positive control (20 microliter) plate
Need the DNA concentration to finish calculating competent cell efficiency
Week 4
5 New modifications with secondary structure- With these modifications we would have expressors that have cDNA of lengths
15 base 3prime truncation, 20 base 3prime truncation, 25 base 3prime truncation, 9 base truncation from BOTH ends, 5 base remaining from 3prime – rehashing of Expressor Naught
SEPTEMBER
Week 1
Interlab: Measured fluorescence using plate reader and finalized protocol for plate reader. (Fluorescein standard curve) calibration and standard curve mode (serial dilutions)
Week 2
Analyzed plate reader data to find that our repressor provided imperfect and incomplete repression
Week 3
Lab meeting with PI to determine the goals for the team and Wiki
Week 4
Helped Lambert High School team with the plate reader
Interlab: filled out the three protocol forms for the plate reader and the Excel data sheets
OCTOBER
Week 1
Write protocol to begin construct tests with AFB1
Week 2
Overnight incubation with culture, bacteria, and different concentrations of toxin
Constructs tested: Ea, E3, E3R, and a blank no bacteria with toxin
Week 3
Adapted previous protocol by applying toxin to media during exponential growth phase in order to stimulate aflatoxin-RNA binding over the competing RNA-RNA binding
Possible signal but further testing is needed for two different conditions (overnight vs. hour aflatoxin introduction)
