Difference between revisions of "Part:BBa K1692024"

Latest revision as of 16:55, 24 September 2015

Autolysis

Note:
Please click here if you're looking for the cipA construct (BBa_K1692027)
Please click here if you're looking for the cipA-mSA construct (BBa_K1692035)

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.

Autolysis Overnight Culture After growing overnight culture of the autolysis and the negative control, Pank+Imp, we measured the OD600 over a period of four hours. The chart showed that the autolysis culture did not exhibit the expected sinusoidal curve from the continual repeat of cell growth and lysis.

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.

Autolysis Growth Rate We compared the growth rate between the autolysis and the negative control. R_auto_avg is the autolysis growth rate and R_Pank_avg is the Pank_Imp growth rate. The growth rate comparison showed no statistical significance between the rate of autolysis and the rate of the negative control.

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