Major IndieBio update, First visible colonies from pASapI-aada algal transformation experiment, and a positive colony PCR product.

When we began the IndieBio EU accelerator program, we started experimental work immediately. With the proinsulin and spectinomycin resistance cassettes successfully constructed by Sebastian, and two additional plasmid constructs in the pipeline, we proceeded with our microalgae work while Sebastian put in the order for the Western blotting reagents to probe for insulin expression in the E. coli.

We began with our first construct which we confirmed was working in E. coli, pASapI-aada.

 

Our first working genetic construct (pASapI-aaDA)

 

We successfully transformed this plasmid into E. coli and miniprepped enough plasmid DNA to begin doing some genetic transformations.

 

 

 

We confirmed that the gene was functional using PCR and an antibiotic assay. We then began with a simple protocol from our scientific advisor, Dr. Saul Purton, who had provided the original vector (pASapI) which we used as the backbone of our genetic constructs. We started with pASapI-aada (antibiotic resistance) because we needed to have a working protocol by the time we were finally ready to start transforming the microalgae with the insulin gene.

 

Based on the work of our scientific advisor, Dr. Saul Purton (left), we wrote our own short protocol for transforming a microalgae with pASapI-aada (right).

 

We used a method of glass bead agitation to introduce the foreign DNA into the microalgae.

Over the span of almost 3 months, we worked around the clock on our experiments while also getting business mentoring through IndieBio and SOSV. 

 

 

 

We were even featured in a few newspapers. We knew that running our business (MicroSynbiotiX), attending the IndieBio mentorship classes, and conducting the experimental work would be very challenging, but we found that we still thrived even under pressure. We worked diligently, from 9am-5pm on most days to complete everything, including coming into the lab on weekends.

 

The index file for our experimental research on Benchling

 

Antonio replating the microalgae cell wall mutant strain onto fresh TAP media

Towards the end of the program, we had found an optimal media composition for algal growth, we assembled two additional plasmids for testing (a blue chromoprotein and a fluorescence gene), we were able reduce or eliminate the contamination that was plaguing our experiments in the first month, and we performed 5 replicate algal transformation procedures using our pASapI-aada plasmid DNA, of which one gave us some colonies that tested positive for the gene.

 

Thanks to Simon Porphy, we found a good media composition (using 3% yeast extract) that gave us strong heterotrophic growth. By around Day 60 of the program, we were getting great biomass concentrations. Because it can take anywhere from 2 to 5 weeks to get any transformed colonies, we did as many replicates as we could until we saw some growth.Eventually, some little green colonies finally reared their heads.

 

In order to confirm whether they were indeed transformed, we did a colony PCR and got positive bands using primers that were complementary to the spectinomycin gene.

 

Our colony PCR results for the colony we discovered from the algal transformation experiments. 

We then took this colony and inoculated it in liquid media with antibiotics. We will perform additional tests to further confirm whether it is indeed a transformed colony, but the results are positive so far. Although the science moves slowly at times, make no mistake that a lot of progress has been completed. Thanks to IndieBio, what would have taken us at least a year to do on our own, was completed in less than 3 months. Our company, MicroSynbiotiX, has secured letters of interest from large aquaculture farms, and formed a strategic partnership with a French pharmaceutical company that specializes in fish vaccine challenge trials (ICTYO Pharma). After we complete the open-source insulin experimental work, we want to use our own expression system to produce algal oral vaccines.

 

A dinner in Cork with some members of the other great IndieBio EU teams also pushing some innovative ideas.

In less than two weeks, the IndieBio accelerator program will come to an end, culminating in a final Day 90 Investor Pitch, held in Cork City, Ireland. If you're in the area, I invite any donors or followers on the experiment.com page to check out the event and grab a ticket to come see all the great work that each team has completed so far (just click the image below). Even if you can't attend in person, the whole show will be live-streamed for free as well and the link can be found below.

 

Immediately after the program, we will continue most of the laboratory work in San Jose, California at BioCurious and in New York Botanics, which is run by Sebastian Cocioba. By January 2017, we hope to secure additional investment after the IndieBio program and set up another laboratory facility. We will still continue working on the algal insulin project and give additional updates as we complete additional milestones. 

Have a great summer, everyone! 

UPDATE:

 

After about 5 weeks, some visible colonies also began to appear on another set of replicate plates. These are also being re-cultured on fresh media and will be PCR genotyped in San Jose, California. A positive PCR result was obtained.