Final Project

Microfluidic system an artificial bioproduction for vaccine antigens

Why simplify vaccine production?

•Expensive

•Bioreactor

•Complex procedures

•Purification of products

•Multiplex Phases

•Antigen production

•Laboratory space

Why multiplexing using synthetic minimal cells?

Optimization for use plasmid

Same reaction

Multiplex Phases

Laboratory space

Difficult procedures

More production

Proposal

The purpose of this study is the multiplexing of artificial free cells to produce proteins in a microfluidic system allowing one hundred reactions in a single process. With this multiplex system, we can obtain up to 10,000ug/ml of protein.

Methods

Gene selection and plasmid (antigen)

In a previous study as a co-investigator, we found antigens with potential protective capacity.

First, we isolated and sequenced a Pasteurella multocida genome (Alpaca pathogen), using Illumina Hiseq2500.

Then we analyzed its genome, for it we used Velvet (assembler of genomes), RAST (annotation of genomes) and then they were uploaded to GenBank.

To determine the antigens we use the databases VFDB and ProtegenDB.

The candidate antigens were then then cloned in the vector pET one hundred two Champion system. The candidates were expressed in E coli and -then tested on model and alpaca animals. The plasmid containing the OmpA candidate was selected for this project.

Project: Reverse Vaccinology: Development of a new generation vaccine for the control and prevention of pasteurellosis pneumonia in Alpacas. 133-FINCyT-IB-2015

ØNational University San Marcos (UNMSM),

ØInstitute Southamerican Camelids (CONOPA),

ØNational Institute of Health Peru – David Tarazona

Synthetic Minimal Cells to microfluidics

Classic reaction

1. Add

10uL reaction

ü(a) 7.5uL of enzyme mix

ü(b) pET-alpaca5 plasmid (5nM) -

ü(c) Tool plasmid (5nM) Extra

ü(d) Water

2. Mix by gently pipetting up and down several times.

3. Incubate at 29-30C for 3-4h.

4. Analyze Bradford and ELISA

The model for synthesis of protein by minimal synthetic cells consists of three events, the first, a four-component mixer that includes transcription enzyme transcripts, plasmid with the gene of interest, extra plasmid (gamma 70) and water. The second, a system of separation of total components to 50 channels of ten microliters each, which will be paused for 3-4 hours to initiate reaction, this is where the production of proteins takes place. Finally, the third one will join the components produced by the 50 channels in a central channel.