Composite

Part:BBa_K611059:Experience

Designed by: Nicholas Csicsery   Group: iGEM11_UC_Davis   (2011-09-27)


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Applications of BBa_K611059

The iGEM Stockholm 2019 team has chosen to characterize this biobrick as part of the bronze criteria. We can activate the expression of tetR by adding different concentrations of arabinose, which will bind to AraC3 and allow for transcription of the following genes. On the other hand, we have ptet promoter variant #9, followed by an RBS and a GFP protein. When tetR binds to ptet promoter, it inhibits the expression of GFP. This composite part is useful for teams that would like to have a gene that can be repressed using arabinose by simply changing the GFP gene with their gene of interest. Our team was able to show the behaviour of this biobrick under different conditions, which can help those teams interested in working with this composite part. For example, we have shown conditions at which the expression of GFP is stable over time.

User Reviews

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iGEM Stockholm 2019

Team iGEM Stockholm 2019 characterized this composite part under different concentrations of arabinose and using two different backbones: the standard backbone used in the distribution kit and a backbone containing AraC under a constitutive promoter. For this purpose, we transform TOP10 E.Coli cells with BBa_K611059 in two different backbones (psB1C3 and AraC-psB1C3) and measured fluorescence over time (5h) and under different concentrations of arabinose (from 1M to 1 microM). Fluorescence was measured using FLUOstar OMEGA microplate reader, from BMG labtech, at a initial OD=0.4. For the whole protocol, please visit iGEM Stockholm 2019 Wiki page

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Image 1: AraC3- BBa_K611059 fluorescence over time and under different concentrations of arabinose (n=2)

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Image 2: comparison between C3-BBa_K611059 and AraC3- BBa_K611059 fluorescence over time and incubated on 1M (n=2)

Our results confirm that fluorescence decreases with higher concentrations of arabinose, so there seems to be a negative correlation. The system seems to reach an equilibrium point around 100µM, were the rate of cell growth-GFP production seems to be equal to the amount of inhibition by pbad-tetR. This result can be useful for systems where an stable production of a protein over time is required. Teams could design their construct by introducing the sequence of their desired protein instead of GFP, therefore, obtaining a stable expression of their protein over time, even if bacteria multiply.

In order to repress the system totally, one should either test for higher concentration of arabinose (more than 1M) or testing the system for a longer time period (longer than 5 hours). One possible outcome is that the pbad promoter is not strong enough to express as much tetR as required to repress the system, so saturation with arabinose will not decrease the expression of GFP. In this case, we recommend future teams that wish to further characterize this system to perform a similar experiment but including different concentrations of anhydrotetracycline (ATc) or its analogue, doxycycline (dox). This inhibitors bind to TetR protein and inhibit its function, this is, TetR can no longer bind to ptet and inhibit the expression of GFP. This would allow the team to understand how much concentration of TetR is necessary to repress the system.

Last, but not least, our system shows increased GFP production when AraC protein is overexpressed, as observed in image 3. AraC protein binds to pBad promoter and in the presence of arabinose, it promotes gene expression. These results seem to correspond to our third hypothesis, and it demonstrates that overexpression of AraC protein is required to achieve higher promoter strenght . Therefore, it is recommended for future teams using this system and who wish to achieve higher production of protein to change the backbone of the biobrick to one containing the AraC gene.

See detailed protocol at iGEM Stockholm 2019 wiki page