Team:Thessaly/Measurement
Measurement
Overview
We consider experimental data to be integral toour contribution in iGEM. This year, we did a comprehensive characterization of our genetic devices, before deciding the final conditions of main experiments. These include evaluating 1) different chemical inputs to our NOT-GATE devices, 2) a vast concentration range for each input, as well as 3) different GFP variants. We then used the gathered data to test the functionality of our NOT-GATE, demonstrating a functional Proof of Conceptwhile we tested our Reporter Module and provided other teams with the right data to adjust our work to their project.
Aim
Our goal was to provide documentation about existing parts and the functionality of new genetic devices and also determine the limits of our design. To do that,we characterized an existing part (BBa_K2924016) that will be available for use to other teams, which also, helped us build a successful proof of concept. Furthermore, we tested the functionality of our Reporter Module, which constitutes an alternative way to transmit signals, using electrochemical reactions. to their project.
Characterization of promoter FliC
Our Prom Module was designed to detect absence of several Short-Chain Fatty Acids (SCFAs)
in the human intestine (Read more). However, previous characterization efforts only
included one SCFA, butyrate, resulting in partial characterization which was inadequate for
us to run experiments. To evaluate the ability of pFliC to detect other SCFAs (Tobe, Nakanishi
and Sugimoto, 2010) and its general behavior in our E. coli strain, we used two SCFAs,
acetate and propionate, at concentrations spanning 5 orders of magnitudes at several time-
points. Moreover, we used three GFP variants to assess promoter activity in E. coli MC1061,
which we used for expression experiments. Finally, we visualized our data that include the
appropriate standards and controls, using detailed graphs that underlie the results of our
experiments.
For more information: Click here
Proof of Concept of our Modules
After characterizing our part, we used the information to conduct our main experiments.
Using different conditions helped us identify the right conditions for optimal singal output, in
order to have successful experiments. In addition, we resolved this situation in a way that
we could have an efficient method of proving the functionality of our Modules. To do so, we
used more than two SCFAs to activate our circuit in a series of time-points that allowed us to
study the nature of our design.
For more information: Click here
Reporter Module
There are a lot of exciting methods to visualize the output of a genetic circuit, but there is
only a limited way to transmit a signal. To further contribute to the competition, we
designed a Reporter Module that is based on a recently published Tyrosine-Tyrosinase
Electrochemical Gene Circuit converting the biological information into electrochemical
signal and achieving the communication between bacterial-based systems and electronic
networks (Vanarsdale et al., 2020). As we wanted to test our Module, we used different
controls to understand the response of this, checking the signal, when we had bacteria with
and without our construct and adding our inducer or not.
For more information: Click here
References
Tobe, T., Nakanishi, N. and Sugimoto, N., 2010. Activation of Motility by Sensing Short-Chain
Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in
Enterohemorrhagic Escherichia coli. Infection and Immunity, 79(3), pp.1016-1024.
Vanarsdale, E., Hörnström, D., Sjöberg, G., Järbur, I., Pitzer, J., Payne, G. F., Van Maris, A. J.
A., & Bentley, W. E. (2020). A coculture based tyrosine-tyrosinase electrochemical gene
circuit for connecting cellular communication with electronic networks. ACS Synthetic
Biology, 9(5), 1117–1128. https://doi.org/10.1021/acssynbio.9b00469
