Part:BBa_K2764002
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
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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Characterisation by Team HK_GTC in 2019
As shown above, the growth rate of bacteria with BBa_K2764002 increases steadily and is likely to be faster than the control, bacteria with GFP with a promoter,BBa_K608002. The two plasmids have similar size but their promoters are different and have different strength.
The control utilised J23104 as its promoter which has a relative strength of 0.72 on the Anderson promoter scale, and BBa_K2764002 utilised promoter J23108 which has a relative strength of 0.5 on the Anderson promoter scale, lower than that of the control.
Comparing the growth rate of bacteria with BBa_K2764002 to the bacteria with GFP control throughout the 8 hours,BBa_K2764002 has a higher growth rate than that of the GFP in terms of the reading of spectrophotometer at OD600 and its percentage increase. As the iGEM18_Hong_Kong_UST mentioned, the toxic protein AlkL can actually import long chain fatty acids and aid cell growth. The protein can boost in cell growth by the import of more and more fatty acids overtime and can offset the negative effect of toxicity.
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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