Difference between revisions of "Part:BBa K2764002"

Revision as of 04:51, 17 October 2018

Alkane channel protein with BBa_J23108 Constitutive promoter

Usage and biology

As shown in literature, the AlkL protein may be toxic and inhibit the growth of its host. We decided to test the severity of this toxicity and compare by using Anderson promoters of varying strength. BBa_K2764002 utilizes the promoter BBa_J23108, which gives the biobrick a medium promoter strength of 0.5 on the Anderson promoter scale. This will be used to measure the effect of toxicity of AlkL based on the growth curve of bacteria transformed with this biobrick. In this experiment, we used a bacteria transformed with a plasmid containing insert of BBa_I13401 as the control. This is because the control's plasmid has a similar size to our part's plasmid.

Growth curve
File:Https://static.igem.org/mediawiki/2018/6/66/T--Hong Kong HKUST--AlkL 002 growth.png

As shown, the growth rate of bacteria with BBa_K2764002 rises steadily, but not as rapidly as BBa_K2764001, and peaks at 0.7AU at T=8

Compared with the growth of the control, bacteria with BBa_K2764002 has slightly lower growth throughout the 8-hour period. This shows that AlkL indeed inhibits growth of the host. Therefore, AlkL is proven to have a toxic effect on the host. However, compared with BBa_K2764003, which has lower promoter strength, BBa_K2764002 has higher growth in the 8-hour period. Supposdly, the more AlkL expressed, the more toxic the effect to the cell is. However, it is hypothesized that AlkL can also import long chain fatty acids contained in the yeast extract from LB. The import of fatty acids may enter β-oxidation directly and aid the growth of the cell instead. As more time passes, more AlkL is expressed and more fatty acid is imported into the cell as a result. The boost in cell growth by the import of long chain fatty acids may offset the toxic effect that inhibits cell growth. Hence, over time, the cell containing BBa_K2764002 may be able to offset the effect of cell growth inhibition. This is supported by the fact that the final absorbance of bacteria with BBa_K2764002 shows a trend of catching up to that of the control over the 8-hour period and it absorbance at T=8 is just slightly below the absorbance of the control. This supports the hypothesis that AlkL can boost cell growth over time as it may import more and more long chain fatty acids over time. Hence, while AlkL is toxic, it can help import fatty acids that slowly offsets the toxic effect.

References:
1. Grant, C., Deszcz, D., Wei, Y., Martínez-Torres, R., Morris, P., Folliard, T., Sreenivasan, R., Ward, J., Dalby, P., Woodley, J. and Baganz, F. (2014). Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes.
2. T. P. Call, M. K. Akhtar, F. Baganz, and C. Grant, “Modulating the import of medium-chain alkanes in E. coli through tuned expression of FadL,” Journal of Biological Engineering, vol. 10, no. 1, May 2016.
3. “Preparation of LB (Luria-Bertani), Miller broth,” Lab Protocols, 02-Dec-2017. [Online]. Available: http://mcblabprotocols.com/protocols/preparation-lb-luria-bertani-miller-broth/. [Accessed: 8-Oct-2018].
4. S. en P.- www.schuttelaar.nl, “How it's made,” How it's made - Yeast extract. [Online]. Available: http://www.yeastextract.info/yeast-extract/how-it-s-made. [Accessed: 8-Oct-2018].
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