Experiment background 04

Dr. Sánchez San Martín found and brought to my attention a paper on synthetic peptides based on segments of the griffithsin protein structure, which would influence the direction of the research would take leading to our experiment. 

 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014360

The scientists had designed, synthesized and explored 3 peptides based on the beta hairpin structure found in native griffithsin, which they named grifonin-1, grifonin-2, and grifonin-3.  Grifonin-1 showed the most powerful anti-viral activity.  

We are constantly referencing this paper.  To our knowledge no one else has designed a DNA sequence to express a peptide structurally similar to the synthetic peptide grifonin-1 in bacteria.   

I back translated the grifonin-1 peptide sequence to a DNA sequence.   Dr. Sánchez San Martín redesigned the amino acids, making substitutions to keep stability in E-Coli and designed the DNA sequence for expression in the vector.  We then chose a his-tagged pET expression plasmid.  With the design complete, we started to work on the protocol for the experiment.  At this point, Dr. Sánchez San Martín started a new job at UCSF, where she has been working on the problem of Zika Virus.  At this point, another scientist, who I will designate as Dr.Y started helping me with the protocol I was writing.  Dr. Y is a pharmacist and neurobiologist. Biomedical scientist Celena Carrillo joined the team and has been going over the protocol with me. The protocol outlines our methods and materials for the experiment and out of that a budget was derived.  

Excerpt from the protocol:

Nenufar – antiviral research project 01 – Expression and purification of peptide LBOUNCER from the synthetic  gene VRBNCR-01 inserted into an e-coli plasmid vector, transformed and induced to grow on bacteria.  Explore how to do this in a fast and consistent way with the highest purity.

Experiment 01A:

DNA synthesis of the VRBNCR-01 gene which is inserted into a pET15 expression plasmid with a His 6 tag on the N-Terminus followed by a thrombin cleavage site.  The gene will be cloned into pET15b via NdeI and BamHI. We will use heat shock to transform into competent cells, then plate out on Amp-agar to grow colonies.   Induce for expression.

Then on to Experiment 01B: purification using chromatography and SDS page

I contacted a lab in the San Francisco Bay Area where we can work.  All in all, we are going to do a lot of improvising so we can stretch the budget and work for at least 6 months on the experiment. Everyone is volunteering their time. We have wonderful mentors and we are learning by doing, the biohacker way.  It is always a steep learning curve as anyone who has worked in molecular biology knows.  The journey of discovery makes it worthwhile.  Thanks to all of you who are making this possible.