Team:Lund/Design/Design
Design
Introduction
Our goal was to co-express VHb with a target protein and study the effect that VHb has on the protein yield at different expression levels. The key idea is that expressing only a small amount of hemoglobin will lead to an insufficient oxygen utilization and consequently non-optimal protein expression. On the other hand, overexpressing VHb should result in a depletion of cell resources as the relative amount of energy gained from the increased metabolism will be overtaken by the amount of protein expressed. The most favorable expression level should therefore lie somewhere in between.
When designing constructs to test the hypothesis with, there are some design specifications that must be considered. The expression level of VHb should be controlled independently of the target protein and should span a wide dynamic range. In addition, it should be easily modulated and easily integrated into already existing systems. Finally, for the project to be of practical relevance, the entire system should be simple, safe and scalable. With these criteria, we now discuss the design of our constructs.
The choice of regulator
There are many ways of regulating expression levels in organisms. It can be done either in an integrated fashion by control on the genetic level, or in an independent manner by control of an external environmental factor, such as the concentration of an inducer, in order to achieve a desired response. Clearly, both ways have their own specific advantages: the first option does not require supervision once the transformation is accomplished, whereas the second allows for a higher level of control during production. For our project we chose the first option since once the specification is accomplished, no further requirements are put on the expression system. This allows the construct to be more easily integrated into other organisms where the inducer might interfere with other processes or be hard to control.
Given the choice of regulating the expression on a genetic level, the next choice that arises is whether the regulation should be in the transcriptional or post transcriptional stage. Methods belonging to the latter class include the modification of ribosome binding sites. Also, one could perform the entire regulation independent of any external circuitry by tuning the codons of the VHb sequence for the specific organism used. However, we opted for the former method by using the set of constitutive Anderson promoters since they are well characterized and often used within the iGEM community. In addition, Imperial college 2014 managed to successfully express VHb under a medium/strong Anderson promoter to increase the production of cellulose. Therefore it seemed natural to build upon their work and put the Anderson promoters to use in a more general setting.
The choice of host
For the host organism, we wanted one that was safe, easy to work with and commonly used in industrial applications. The options we mainly considered were Escherichia coli and species of yeast such as Pichia pastoris or Saccharomyces cerevisiae. While yeast can be a better choice for production of certain proteins, e.g. those that require extensive post-translational modification, we decided to use E. coli as it is easier to work with, grows faster and is commonly used in industry. In addition, VHb is a bacterial protein. We chose the strain BL21 for expression, and for plasmid propagations we tried both TG1 and DH5α.
Bringing it all together
Fig. 1 illustrates the final construct of our VHb biobrick. It consists of an Anderson promoter and a ribosome binding site followed by the vitreoscilla hemoglobin gene (vgb), and ends with a double terminator sequence. The Anderson promoter was chosen to be variable to control the vgb expression level. The ribosome binding site used was BBa_B0034, which is of medium strength and one of the most used parts in the registry. Finally, we opted for two terminators to minimize the risk of leakage.
