Difference between revisions of "Team:Lund/Design"

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          <h3 class="section-heading">Overview</h3>
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            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 this works or for a short review of the scientific background, see the <a href="">theory</a> section. To find out more about how we have designed the biology and experiments of our project, see the <a href="">design</a> and <a href="">experiments</a> section respectively.
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           <h3 class="section-heading">Project idea</h3>
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            As described in the <a href="">project description</a>, one major bottleneck in the microbial production of many bioproducts is the amount of oxygen that is able to dissolve into 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.
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           <p>Our goal is 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 will 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.</p>
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           <p>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. </p>
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            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 sources and consequently produce more proteins under low oxygen conditions.
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            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, more valuable protein and expressing too little will leave the benefits underutilized. As illustrated in fig. 1, the best level of expression should be somewhere in between.
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            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 expressed the protein at different levels. In this way, one is able to both harness and fine tune the benefits of VHb.
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          <p> There are many ways of regulating expression levels in organisms. It can be either done in an integrated fashion by control on the genetic level, or in an independent manner where one controls an external environmental factor, such as an inducer concentration, 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. </p>
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           <p> Given the choice of regulating the expression in a genetic level, the choice that arises is whether to do this in the transcriptional or post transcriptional stage. Methods belonging to the latter class includes 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 one 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 co-express vgb 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.</p>
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            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 pVHB1, pVHB2 and pVHB3. The plasmids are then screened for the best yield, where the best performing vector is further tested for upscaling. Once accomplished, the plasmid is ready to be used for its final intended application.
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          <p>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, 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 native for bacteria. We chose the strain BL21 for expression, and for plasmid propagations we tried both TG1 and DH5α. </p>
 
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          <h3 class="section-heading">Bringing it all together</h3>
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          <p>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 gene, and ends with a double terminator sequence. The Anderson promoter was chosen to be variable to control the level of expressed vgb. 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.</p>
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          <p>Fig. 2 illustrates an example of how the VHb system can be incorporated with a target protein of interest. By appending the biobrick containing the target protein downstream of the VHb biobrick, it is possible to express both at the same time. Consequently, by making an assay and screening out a span of Anderson promoters with varying strengths, one can fine tune the expression level for the specific protein. Once this is accomplished no further modifications need to be performed and the system can be considered set and ready to be used.</p>
 
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            <figcaption>Figure 1: The approach.</figcaption>
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Revision as of 17:30, 11 October 2018

Design

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 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 is able to dissolve into 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 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 sources 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, more valuable protein and expressing too little will leave the benefits underutilized. As illustrated in fig. 1, the best 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 expressed the protein at different levels. In this way, one is able to both harness and fine tune the benefits of VHb.

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 pVHB1, pVHB2 and pVHB3. The plasmids are then screened for the best yield, where the best performing vector is further tested for upscaling. Once accomplished, the plasmid is ready to be used for its final intended application.

Figure 1: The approach.
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