Team:TP CC-SanDiego/Results.html


iGEM San Diego

PCR Amplification
Amplifying vectors and synthesized genes to have desirable tails.

By adjusting PCR settings, we were able to amplify the vector and inserts successfully. Successfully amplified inserts and vectors with the desired ends were used for Seamless Cloning.

After seamless cloning; E. Coli uptake of assembled plasmid

For AA, AZ, BA, and BZ, at least 10 colonies were grown for each LB+Amp agar plate that cultured the four different types of transformations.

(Note: AA refers to alpha-amylase-ADTZ, AZ refers to alpha-amylase-ZHD101, BA refers to beta-lactamase-ADTZ, and BZ refers to beta-lactamase-ZHD101.)

Colony PCR
PCR of colonies as template DNA

After the transformation in an LB+Amp agar plate, numerous colonies were selected individually for Colony PCR. The columns with the bands superficially signifies that the desired plasmids were constructed. The colonies that were reported positive were plated separately and re-verified.

AA: 2, 6, 13, 14, 19 || AZ: 1, 2, 4, 5, 6, 7 || BA: 6, 7, 8 || BZ: 2, 3, 5, 6, 7

Sequence Verification
Sequenced colonies vs. Theoretical plasmid

Using NCBI's Align BLAST, the sequencing data was compared with the theoretical plasmid data. If there was a 100% match, we declared these colonies to be successful. All the mutations that occurred in our set of colonies were deletion or addition mutations. AA13, AA14, AA19, AZ1, AZ5, AZ6, and AZ7 were 100% congruent. AA2 was determined to be a very likely because the error occurred in the region where the sequencing data had various "N"'s and the ab1 peaks were fluctuant.

None of the beta-lactamase-attached colonies seemed to have the correct plasmids due to point mutations in the ORF.
AA: 2 (possible candidate), 6, 13 (100%), 14 (100%), 19 (100%) || AZ: 1 (100%), 2, 4, 5 (100%), 6 (100%), 7 (100%) || BA: 6, 7, 8 || BZ: 2, 3, 5, 6, 7

SDS-PAGE gel 1
Visualization of presence and size of protein of interest

We tested four parameters : pellet, supernatant, IPTG-induced, and non-induced. For both pellets and supbernatants, IPTG-induced was hypothesized to have a stronger band for SDS-PAGE, with visible difference in both the pellet and the supernatant. The first gel did show sharp difference between pellet and supernatant, but did not show a clear difference between IPTG-induced and non-induced. It implies that the plasmid was neither expressed nor secreted.

Western Blot
Visualization of presence and size of protein of interest

The Western Blot of two AZ colonies and two AA colonies were studied, both IPTG-induced and non-induced. The bands show that the plasmid was expressed at the very least, since it showed a stronger band in the presence of IPTG in the pellet. However, for the supernatant no bands were visible for neither IPTG-induced, nor non-induced, which shows poor secretion. Possible explanations for this results include:
1. The construct and the attached signal peptides are not functional and do not secrete.
2. E. Coli's secretion system is generally less effective.
3. The proteins are stored in the periplasm and not excreted to the extracellular space.
4. The protein's structure (size or chemical properties) somehow interfered with the ability of it to be transported out.

SDS-PAGE gel 2
Visualization of presence and size of protein of interest

The SDS-PAGE was tried again. This time, only IPTG-induced supernatant was taken into account to compare amongst colonies and narrow down the list of colonies needing to be tested. Some colonies showed expression of proteins of the approximately accurate size in the supernatant, while the previous gel showed none. The ones that did were selected, and of those, a western blot of both IPTG-induced and non-induced was suggested.

Conclusion And Future Directions
The fight will continue.

Two of the four final constructs, AA and AZ, were sequence verified 100%. The SDS-PAGE analysis initially showed inconclusive levels of expression and extracellular secretion of the alpha-amylase attached proteins. The Western Blot, however, confirmed that expression is present, but did not show any signs of secretion. A secondary SDS-PAGE was done for more induced colonies, and there were signs of secreted protein that are to be explored. Future directions include:

1. Successful colonies from SDS-PAGE Gel 2 need to be tested further with Western blotting and compared amongst pellet, supernatant, induced, and non-induced.
2. Try to succeed in the construction of beta-lactamase signal peptide containing recombinant plasmids, since we were not able to for now. Then compare alpha-amylase signal peptide and beta-lactamase signal peptide levels of secretion.

3. Do protein functionality assay of secreted ADTZ and ZHD with HPLC.
4. Try other signal peptides, such as the TAT-dependent pathways or SRP-dependent pathways.
5. Genetically modify plants directly instead of modifying E. Coli.
6. Try organisms with more pronounced secretion systems such as yeasts and fungi.