Part:BBa_K4006002
MT
Background
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.
Cloning into E. coli and Verification
We were able to clone this construct into our plasmid, pASapI, using Gibson assembly and select for transformed E. coli colonies.
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.
Transformation into Chlamydomonas reinhardtii
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
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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
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Arsenic Plot at 50 ppb
Arsenic Plot at 500 ppb
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 40
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 224
Illegal SapI.rc site found at 31
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