Difference between revisions of "Part:BBa K4006004"

Revision as of 20:52, 20 October 2021

MT-6XHis

This part contained a protein coding sequence for metallothionein that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii as well as a 6X-histidine tag for visualization of protein expression in a Western blot. Metallothionein is a metal binding protein rich in cysteine that is commonly produced in bacteria and prokaryotes. It has been introduced to the Chlamydomonas genome before and significantly improves the metal binding capacity at low metal concentrations. This version of metallothionein is codon optimized for use in the C. reinhardtii chloroplast and is an improved part, based off of the previously characterized part, BBa K3275000, human metallothionein from team iGEM19_RHIT. The polyhistidine tag is well established and has been used previously in C. reinhardtii to tag the photosystem I complex.

We were able to clone this construct into our plasmid, pASapI, using Golden Gate assembly 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

Finally, we performed a Western blot to identify the expression of this protein using the polyhistidine tag. INFORMATION HERE.

PICTURES OF BLOTS.

Sequence and Features