Difference between revisions of "Part:BBa K4006005"

Revision as of 20:55, 20 October 2021

arsC-HA

This part contained a protein coding sequence for arsenate reductase that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii as well as a human influenza hemagglutinin (HA) tag for visualization of protein expression in a Western blot. Arsenate reductase catalyzes the reduction of arsenate [As(V)] to arsenite [As(III)]. Using 2 µM arsenate at a substrate, the Kcat of the protein has been recorded as 4.5 min(-1).

We were able to clone this construct into our plasmid and select for transformed E. coli colonies.

PLATE PICTURES

Digestion of the miniprepped DNA in the plasmid pASapI with XbaI and BstXI should result in two bands of approximate sizes SIZE bp and SIZE bp as compared to the original plasmid which should have three bands of sizes 4446, 1376, and 800 bp. Each of the colonies were successfully cloned.

GEL PICTURES

We were able to successfully transform this construct into C. reinhardtii using biolistic transformation. First, we used SOMETHING ABOUT LIGHT, EMMA?? to confirm that the photosystem II subunit had been restored with our rescue plasmid.

RESULTS OF LIGHT THING

Next, we performed PCR using primers outside of the region where our protein was meant to integrate into the genome. JARED SOMETHING ABOUT RESULTS.

IMAGES

The HA tag is also generally well established and has been used previously in C. reinhardtii to tag putative flagellar proteins. Interestingly, though, when we used anti-HA antibodies in a Western blot for our transformant colony's genomic DNA, we visualized two distinct bands present throughout all of our constructs and the wildtype. They were not the right size for the arsC, yet we have seen no literature reporting on the natural presence of the HA sequence within the C. reinhardtii genome.

PICTURES OF BLOTS.

Sequence and Features