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)