Part:BBa_K1692024
Autolysis
Note:
Please click here if you're looking for the cipA construct (BBa_K1692027)
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We create an autolysis system based on the concept of quorum sensing. The idea of quorum sensing is that Gram-negative bacteria releases a special molecule known as [N-]acyl-homoserine lactones (AHL) that can bind to the luxR proteins. The complex formed from using 2 AHL molecules with 2 luxR proteins can then bind the luxpR promoter and activate any downstream gene [1]. Our construct combines the part from 2010 Tokyo NoKoGen (BBa_K317039) and our ptet-luxl generator construct (BBa_K1692023). The specific AHL molecule used is 3OC6HSL, which is found in V. fischer . Our goal is to be create an automatic means of self-lysis once the cells reach a certain cell density.
Functionality Test
We successfully sequenced the construct and did some preliminary testing with the lysis device. In our preliminary test, we let the cells grew for a few hours before starting measuring the OD600 per hour. We tested three biological replicates of our autolysis samples and compared those to three replicates of our negative control, which we chose to be the Pank-Imperial strain (BBa_K1692021). The data did not show that the autolysis function properly as we expected that the OD600 would oscillate in a sinusoidal fashion as cells grow and lyse.
We thought that perhaps the autolysis device might have slower rate of growth than the negative control; thus we graphed the growth rate over time.
Unfortunately, the growth rate over time did not show that there was a statistical significance between the autolysis samples and the negative control. We believed that the experiment can be better designed by centrifuging the cells after every hour to remove the supernatant (containing the dead cells) before doing the OD600 measurement. It is possible that the cell debris could affect the OD600 measurement, preventing an accurate determination of cell density over time. Alternatively, we plan to add a GFP gene within the construct such that we can use fluorometer instead to measure the GFP level over time. Since GFP would be expressed whenever the lysis gene is expressed, the level of GFP can help to accurately if there's a sinusoidal relationship between cell viability over time for the autolysis system.
Conclusion
In conclusion, we have created and sequenced an autolysis device. Though our preliminary data did not show promising result, we believed that adjustment to the experimental design can provide us with a more accurate conclusion to the functionality of the autolysis system. We envision autolysis to have many practical applications and one such application is combining autolysis with our designed P(3HB) part (BBa_K1692021) as a means of releasing plastic into the media without having to do extraction process.
References
[1]. Waters, Christopher, and Bassler, Bonnie. "Quorum Sensing: Cell-to-Cell Communication in Bacteria." Annu. Rev. Cell Dev. Biol. 2005 (21): 319–46. Print.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 875
Illegal NheI site found at 898
Illegal NheI site found at 3268
Illegal NheI site found at 3291 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 717
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2873
Illegal AgeI site found at 2943 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1853
Illegal SapI site found at 3524
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