Making Inexpensive Insulin Using Microalgae

Methods

Summary

please check out the basic overviews of each cloning experiment we're planning to conduct so far. Additional information about how things work can be found here, and here.

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Cloning of Spectinomycin Resistance Gene (SmR, also annotated as aaDa) into the pASapI Chloroplast Expression Vector. (Completed April 2016)

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Cloning of Proinsulin Coding DNA Sequence into the pASapI Chloroplast Expression Vector. (Completed April 2016)

Cloning of teal chromoprotein into pASapI Chloroplast Expression Vector (Completed)

Cloning of Algal Cyan Fluorescent Protein into pASapI Chloroplast Expression Vector (Completed)

Verification of pASapI-aaDa expression in E. coli (Complete)

Transformation of pASapI Genetic Constructs into Chlamydomonas reinhardtii (Complete)

Update: First preliminary transformed algal colonies observed for pASapI-aaDa. Colonies survived reculturing on selective antibiotic media.

Update: First Preliminary transformed algal colonies observed for pASapI-CFP and pASapI-teal.

Challenges

We initially experienced some challenges in our cloning experiments. We theorized that the issue was most likely due to secondary structures preventing our gene from being ligated properly. We overcame this problem by increasing the ligation time to 36 hours. The proinsulin bacterial work has also been delayed due to issues with getting the Western Blotting reagents shipped on time. Although the materials are now here, we had delayed some of the E. coli insulin work in order to complete more microalgae transformation work during the IndieBio EU three-month program. In addition, microalgae grow extremely slowly. We did not observe our first transformed colonies until 5 weeks after our first transformation experiments. 

Pre Analysis Plan

All molecular cloning results are verified using both PCR and sequencing.