Overview

In this section we describe the approach we have taken in our project. We start by explaining the what, why and how of our project and proceed by describing an intended user scenario. To read about the theoretical details, such as how this works or for a short review of the scientific background, see the theory section. To find out more about how we have designed the biology and experiments of our project, see the design and experiments section respectively.

Project idea

As described in the project description, one major bottleneck in the microbial production of many bioproducts is the amount of oxygen that can be dissolved in the growth medium. Clearly, this issue requires no more explanation than that for any organization, the product of interest must be produced in sufficient quantities in order to be practical, be it enzymes, biofuels or pharmaceuticals. The issue of low expression proteins is a big problem in biopharmaceutical research as confirmed by both AstraZeneca and NovoNordisk.

The approach we have taken to combat this is independent of any environmental variables. By modifying the organisms to co-express Vitreoscilla hemoglobin (VHb) along with the desired product, the oxygen uptake is increased. This allows the cells to utilize more carbon and consequently produce more proteins under low oxygen conditions.

However, with this approach, a problem arises. How much hemoglobin should be co-expressed? Obviously, expressing too much will deplete the cells resources to produce the other valuable protein, and expressing too little will leave the benefits underutilized. As illustrated in fig. 1, the optimal level of expression should be somewhere in between.

We are tackling this additional issue by directly leveraging the power of synthetic biology. By utilizing the library of constitutive Anderson promoters, a set of inserts encoding VHb can be created which expresses the protein at different levels. In this way, the benefits of VHb can be both harnessed and fine-tuned.

Scenario

A hypothetical user scenario using our project is illustrated in fig. 2. Given a protein of interest, the first step is to create a library of vectors containing both the gene encoding the target protein and VHb, with the latter expressed under various promoter strengths. This is illustrated by the upper part of the figure where the plasmids are denoted plasmid₁, plasmid₂ and plasmid₃. The plasmids are then screened for the highest yield and/or productivity, where the best-performing vector is further tested for upscaling. Once accomplished, the plasmid is ready to be used for its final intended application.