Difference between revisions of "Part:BBa K4006002"

Revision as of 18:25, 21 October 2021

MT

This part contains a protein coding sequence for metallothionein that has been codon optimized for use in the chloroplast of Chlamydomonas reinhardtii. 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.

We were able to clone this construct into our plasmid, pASapI, using Gibson assembly and select for transformed E. coli colonies.

Negative Plate: Positive Plate:

Digestion of the miniprepped DNA in the plasmid pASapI with XbaI and BstXI should result in two bands of approximate sizes 4446 bp and 2332 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.

We were able to successfully transform this construct into C. reinhardtii and test integration, localizaton, and effect on arsenic uptake in the algae.

Testing for Integration of Plasmid via Fluorescence with Spectrophotometry

WRITEUP

Line

Bar

Testing for Localization of Plasmid via PCR

PCR was run on extracted Chlamy genomic DNA from the successfully transformed construct to confirm the integration of the rescue genes and recombinant proteins. This was performed to confirm integration in the chloroplast genome, as the rescue system is active when integrated into the chloroplast genome. We designed primers according to the deletion strain (CC-4388) sequence. Theoretically, these primers are capable of binding to the rescue gene flanking sites approximately 50bp upstream and downstream of the integration site. This can effectively amplify the site which will confirm that the plasmid donor DNA has effectively been integrated, and that the insertion is of approximately accurate size. Attempts with multiple polymerases and annealing temperatures proved unsuccessful at targeting the region of interest, indicating that the likely problem resides in the effective binding accuracy of the primers. Further review confirmed that the sequence targeted may have slight adjustments based on a current review of genomic sequencing. While we were able to effectively extract genomic information from the wild type strain and this construct, we are still confirming the integration of our insertions for the final constructs. In the image below, MT is represented as C1.

Testing for Increased Arsenic Uptake Capabilities

WRITEUP

Arsenic Plot at 50 ppb

Arsenic Plot at 500 ppb

Sequence and Features