Results

Overview

To accomplish our project objective several recombinant proteins must be produced in order to analyze their effects on a co culture of fibroblasts (L929) and mesenchymal cells after an in vitro burn assay. To evaluate the efficiency of the treatment, a measurement of the proliferation rate was performed using LDH (lactate dehydrogenase) as an analysis metabolite. Given that our treatment involves the usage of a growth factor, the rate at which this protein is released into the medium is critical to avoid adverse effects in the cellular line (such as cancer), for that reason a nano encapsulation with chitosan was also performed to control the rate at which our growth factor is released.

Summary

What did we accomplish?

Nanoencapsulation in chitosan of leptin, BSA and RFP.
Realization of in vitro burn assay.
Realization of leptin proliferation essay.
Obtaining parts with adequate enzyme recognition sites.
Protein production of leptin and tenascin C (More validations are needed).
Co-culture of fibroblast and mesenchymal cells.
Cellular growth in TaCO-BOB hardware.

What happened?

We started ligating parts, no bands of expected size were observed but we attributed this fact to low sensitivity of agarose gels, so we proceeded with transformation and protein induction. Nevertheless, we realized that consistent results were not achieved (bad protein migration rates, sometimes no induction band could be observed). [Unfortunately, we invested a lot of time on this period]
Then we started thinking that our parts did not had the necessary extra nucleotides for proper cleavage of restriction enzymes and what we were truly observing was the effect of star activity and inappropriate ligation, resulting in non specific plasmids which did not contained our whole part but conferred the bacteria antibiotic resistance.

How did we solved it?

We designed primers that incorporate the necessary nucleotides into our parts. Thus, we started having proper migrations of project parts and posterious transformations and protein production of BBLEP.

How should we improve?

By including better protein reporters for the screening of transformed, functional bacteria colonies.