Team:Austin UTexas/Description

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Description

Project Description and Inspiration

Investigating the evolutionary stability of engineered DNA sequences is necessary for using genetically modified bacteria for real world applications. When a construct is added to a cell, resources are allocated towards expression, primarily transcription and translation, of the construct. This creates an additional burden for the cell, making bacterial populations containing the construct less fit than the wild type. Over several generations, cells can accumulate mutations within the genetic construct, rendering it functionless and thereby freeing up more cellular resources towards essential functions. These mutations increase the cell’s fitness and prompt the mutation to eventually sweep the bacterial population. Maintenance of constructs is generally taxing for a cell. Therefore, it is difficult to keep engineered constructs expressible within bacterial populations because cells often cannot support the additional burden associated with the construct for a sustained number of generations. Understanding the evolutionary stability associated with genetic parts will help synthetic biologists create more reliable constructs for long term use in bacterial cell populations.

Figure 1. Loss of function mutants in genetic circuits quickly outgrow their intact competitors.

In 2015, Ceroni and colleagues developed a capacity monitor, a constitutive GFP cassette placed in the genomic DNA of E. coli [1]. Thus, changes in GFP expression indicate changes in the resources available for the expression of GFP. Moreover, capacity is primarily assumed to be the amount of free ribosomes available for GFP expression, which can be quantified through the change in GFP expression over time per cell [2]. If a construct does not exceed the cell's capacity (ergo does not require a lot of the cell’s resources), then GFP expression should be high because cell resources are still available for GFP expression. However, if the construct does exceed the cell's capacity and therefore create burden, the GFP expression should be low, as the cell does not have enough resources left to produce GFP. GFP expression can also be affected by constructs that produce toxins or affect metabolic pathways, but this can be accounted for.

Figure 2. Burden monitor GFP expression decreases when a construct is added to the cell.

The main limitation with any synthetic construct is that its burden may hamper it from continuous expression throughout multiple generations. Developing an understanding of that genetic stability in synthetic constructs is required to maintain the integrity of real-world synthetic biology applications. Therefore, the goal of our project is to measure the growth rates of cells containing genetic circuits to identify burdensome parts, which will therefore allow research done in the future to become more efficient and reliable as well as beneficial to furthering overall part characterization. To do this we used the Ellis Lab’s “burden monitor” design for E. coli to measure the burden of various BioBricks from the iGEM Registry of Standard Biological Parts [1]. Genomic GFP expression levels are monitored as a function of growth rate to determine whether the burden caused by the genetic parts is due to a reallocation of ribosomal resources. We transformed 497 parts from the iGEM Registry into DH10B E. coli containing the burden monitor and assayed the growth rate and GFP expression rate of 330 of these parts. These measurements were then run through our R pipeline to (1) identify burdensome parts by observing percent reductions in growth rate and (2) determine the type of burden created by each BioBrick using genomic GFP expression.

References

[1] Ceroni, F., Algar, R., Stan, G., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature Methods, 12(5), 415-418. doi: http://dx.doi.org/10.1038/nmeth.3339

[2] Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z., & Hwa, T. (2010). Interdependence of Cell Growth and Gene Expression: Origins and Consequences. Science,330(6007), 1099-1102. doi:10.1126/science.1192588