From 2012.igem.org

Revision as of 08:49, 3 October 2012

Note from Dr. Haynes: Hi Nisarg

Please add an outline of the experiments-in-progress on the "Data" page. For instance…

Experiment 1
Aim: To determine that the split Beta-gal part of the biosensor works
Approach: Co-express different versions of the alpha and omega fragments in E. coli
Expected Results: If it works as expected, alpha and omega pairs will give a blue phenotype, and single units (negative controls) will not
Lead experimenter: Abhi
Progress: (to be filled in by Abhi)

Experiment 2
Aim: To quantitate Topo enzyme DNA nicking using a crazy sequencing approach

Etc….

Data

ssDNA Probe Design

Topoisomerase-based DNA Biosensor

insert diagram here

Data For Our New Favorite Parts

D168A Double Cysteine Mutant of Smallpox Topoisomerase, BBa_K891234 - This mutant version of topoisomerase recognizes the YCCTT motif in dsDNA. It cleaves after the last T in this motif, making a single stranded nick, and covalently binds to the 3' phosphate on that thymine.

GFPT1, BBa_K891000 - This part should be paired with GFPT2. This part codes for a 20bp sequence that is complementary to a portion of the genomic GFP coding sequence in E.coli Keio strains.

GFPT2, BBa_K891999 - This part should be paired with GFPT1. This part codes for a 20bp sequence that is complementary to a portion of the genomic GFP coding sequence that comes after the GFPT1 binding site in E.coli Keio strains.

DNA Biosensor

Split Beta-Galactosidase Complementation

Tested alpha fragment of beta-galactosidase for complementation with the omega fragment in vivo. A construct consisting of Streptavadin-Linker-Alpha fragment was transformed into BL21(DE3) E. coli cells that naturally express the omega fragment of beta-galactosidase. Quadrant streak plate in the presence of X-gal produced dark blue colonies. These results illustrate alpha-omega complementation in vivo. In vivo complementation indicates the ability of the two fragments to fuse into a functional beta-galactosidase unit, indicating that the split beta-galactosidase reporter system module of the biosensor was constructed and can be implemented successfully.

Notably, our data shows that the alpha fragment of beta-galactosidase was still able to complementarily bind to the omega fragment and produce a functional unit while linked to streptavidin, a toxic protein. This indicates that the split beta-galactosidase reporter system can still be produced under harsh conditions and within a fusion protein construct. This parallels the conditions that we expect our probe to mature in, given that the beta-galactosidase fragments will also be fused to topoisomerase, which is also a toxic protein that binds DNA. This acts as a proof-of-concept for the DNA-based biosensor, given that both modules of the final biosensor design work as expected.

After 24 Hours

After 48 Hours

Current Research

Current testing with the split beta-galactosidase system includes time-interval testing of colorimetric response, including quantitative measurements of beta-galactosidase concentration over time, omega fragment negative control testing, and in vitro testing of the alpha and omega fragments linked to streptavadin and Magainin.