Part:BBa_K2764001
Alkane channel protein with BBa_J23104 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_K2764001 utilizes the promoter BBa_J23104, which gives the biobrick a strong promoter strength of 0.7 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
As shown, the growth rate of bacteria with BBa_K2764001 rises steadily throughout the 8-hour period and peaks at 0.9AU at T=8
Compared with the control's growth, it can be seen that bacteria with BBa_K2764001 has slower growth from T=0 to T=3. But after T=3, the growth of bacteria with BBa_K2764001 is faster than that of the control. This shows AlkL has an undeniable effect on cell growth. The lower growth of bacteria with BBa_K2764001 observed from T=0 to T=3 proves that AlkL indeed inhibits growth to a certain extent. However, after T=3, the higher growth of bacteria with BBa_K2764001 compared with the control defies expectations. It is hypothesized that while AlkL is toxic to the cell, it also inadvertently imports 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. After T=3, it is possible the boost in cell growth facilitated by the import of fatty acids can offset the inhibition of cell growth caused by the toxic effect of AlkL. As BBa_K2764001 has a high promoter strength, it may be that more and more fatty acids are imported into the cell over time and that boosts the growth of bacteria with BBa_K2764001 such that it can grow faster than the control. For the same reason, bacteria with BBa_K2764001 also grows faster than that of bacteria containing BBa_K2764002 and BBa_K2764003 respectively.
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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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 712
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 364
Illegal NgoMIV site found at 577 - 1000COMPATIBLE WITH RFC[1000]
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