Part:BBa_K2764003
Alkane channel protein with BBa_J23110 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, bacteria with BBa_K2764003 remains consistently low, eventually reaching 0.05AU at T=8 despite a jump at T=2 where it reached 0.1AU. However, the overall growth rate of BBa_K2764003 is still much lower than that of the other 2 parts.
Compared with the growth of the control, bacteria with BBa_K2764003 has much lower growth throughout the 8-hour period. This confirms that AlkL indeed has an effect on cell growth and causes negative growth. This proves the toxic effect of AlkL. In addition, it is seen that up until T=2, there is actually an increase in AU, showing that the cell has positive growth. However, it drops drastically afterwards. This may be because before T=2, not enough AlkL has been expressed to impose severe enough toxic effect to stunt growth. After that, the amount of AlkL is sufficient to pose a dramatic toxic effect that causes negative growth. This is supported by the fact that the absorbance of bacteria with BBa_K2764003 from T=0 to T=1 is similar to that of bacteria with BBa_K2764001 and BBa_K2764002 respectively: they are all collectively lower than the control's growth and exhibit siilar cell density, showing that from T=0 to T=2, AlkL stunts growth but not to the extent that it causes negative growth.
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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