Difference between revisions of "Part:BBa K1894000"

Latest revision as of 20:43, 30 October 2016

Modified GvpA1 coding region

The gene GvpA1 can express GvpA protein, which acts as the main structural protein of gas vesicle in Microcystis aeruginosa. We mutate GvpA1 gene in order to limit the level of GvpA1 expression and change the conformation of gas vesicle protein---- a possible strategy to disrupt the distribution of microcysis in water and control algae bloom.By changing two base pairs, we mutate a serine to an alanine (hydrophilic amino acid to hydrophobic amino acid) and an alanine to a glycin.

Usage and Biology

Gas vesicle protein, which widely exists in all forms of planktonic cyanobacteria, provides cells with buoyancy by changing the density of cells and regulates their vertical distributions in natural waters, enabling them to achieve ideal vertical position in water for growth and subsequent niche colonization. GvpA protein forms the skeleton of gas vesicle and acts as the main structural protein. Therefore, we plans to mutate GvpA1 gene in order to limit the level of GvpA1 expression and change the conformation of gas vesicle protein---- a possible strategy to disrupt the distribution of microcysis in water and control algae bloom. By changing two base pairs, we mutate a serine to an alanine (hydrophilic amino acid to hydrophobic amino acid) and an alanine to a glycin.

Characterization of part BBa_K1894000

The goal of the experiment is to prove that the modified GvpA1 gene will cause limitation of the level of GvpA protein expression. We investigate it by using western blotting and SDS Page in microcysis after the plasmid containing the modified GvpA1 gene has been introduced.

M1: Protein marker
M2: Western marker
PC1: BSA(1 μg)
PC2: BSA(2 μg)
NC: Non-induced whole cell lysing reagent
1: whole cell lysing reagent induced for 16h in 15°C
2: whole cell lysing reagent induced for 4h in 37°C
NC1: Supernatant of non-induced lysis buffer
NC2: Precipitation of non-induced lysis buffer
3: Supernatant of lysis buffer induced for 16h in 15°C
4: Precipitation of lysis buffer induced for 15h in 15°C
5: Supernatant of lysis buffer induced for 4h in 37°C
6: Precipitation of lysis buffer induced for 4h in 37°C

Western blotting result verifies that with microcystis transformed by recombinant plasmid containing modified GvpA1 gene, GvpA1 gene has limited level of protein expression.

Method

Apparatus: Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane（Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.

• Cell extracts were prepared by homogenizing cells or tissues in the lysis buffer (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 45 min.
• The soluble protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad).
• The lysates (50 lg) were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS–PAGE) and transferred to PVDF membranes for immunoblotting assay.
• The membrane was blocked in 5% fat-free dry milk, and probed with antibodies against the interest proteins.
• The blots were visualized using the enhanced ECL reagent.
• The levels of protein expression were semi-quantified by optical densitometry using Image J software.