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Revision as of 00:29, 21 June 2014
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.)
PCR of colonies as template DNA
After transformation in an LB+Amp agar plate, numerous colonies were selected individually for Colony PCR. The columns with the bands superficially signify that the desired plasmids are constructed. The colonies that are reported positive are plated separately and re-verified.
AZ: 1, 2, 4, 5, 6, 7
BA: 6, 7, 8
BZ: 2, 3, 5, 6, 7
Sequenced colonies vs. Theoretical plasmid
Using NCBI's Align BLAST, the sequencing data is compared with the theoretical plasmid data. If there is an 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-lactamse-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
Visualization of presence and size of protein of interest.
We tested four parameters : pellet, supernatant, IPTG-induced, and non-induced. For both pellets and supernatants, IPTG-induced and IPTG-noninduced is hypothesized to have a stronger band for IPTG-induced, with visible difference in both the pellet and the supernatant. The first gel does show sharp difference between pellet and supernatant, but does not show a clear difference between IPTG-induced and non-induced. It implies that the plasmid is neither expressed nor secreted.
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, in the presence of IPTG, and in contrast to non-induced, but only in the pellet. It 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.
Visualization of presence and size of protein of interest.
The SDS-PAGE is tried again. This time, only IPTG-induced supernatant is taken into account to compare amongst colonies and narrow down the list of colonies needing to be tested. Some colonies show expression of proteins of the approximately accurate size in the supernatant, while the previous gel showed none. The ones that do are selected, and of those, a western blot of both IPTG-induced and non-induced is suggested.
The fight will continue.
1. The colonies selected from SDS-PAGE Gel 2 need to be tested further with Western blotting and compared amongst pellet, supernatant, IPTG-induced, and non-induced. .Just one western blotting of two random colonies does not allow for conclusive results.
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-lactamse 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.
