Difference between revisions of "Part:BBa K4006005"

 
Line 1: Line 1:
 +
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.
 +
 +
PICTURE OF PLATES.
 +
 +
Digestion of the miniprepped DNA with X and Y should result in X bands of approximate sizes X and Y.
 +
 +
PICTURE OF GELS.
 +
 +
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.
 +
 +
PICTURE OF WESTERN BLOT.

Revision as of 18:39, 20 October 2021

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.

PICTURE OF PLATES.

Digestion of the miniprepped DNA with X and Y should result in X bands of approximate sizes X and Y.

PICTURE OF GELS.

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.

PICTURE OF WESTERN BLOT.