Team:Hong Kong HKUST/Results
AlkL characterization
Effect of AlkL on cell growth
As shown in literature, the AlkL protein may be toxic when overexpressed and inhibits the growth of its host. We decided to test the severity of this toxicity and compare by using Anderson promoters of varying strength. We chose a gradient of expression strength, ranging from strong (0.7 measured strength) to intermediate (0.5 measured strength), to weak (0.3 measured strength) for this experiment. As a control for comparison of growth rate, we chose E. coli DH5A that was transformed with a plasmid of similar size to our parts plasmid. This is because we wanted to ensure plasmid size was not the factor that limited growth of the host. This plasmid in question contained BBa_I13401 as the insert, which had only 27 extra bp than our alkL-containing plasmids.
The 4 bacterial samples, including 3 parts and 1 control, involved in this growth curve experiment were first inoculated overnight in LB. Then, they were diluted by 100 times in a conical flask and put in a shaker at 37C for an 8 hour period. A sample of 100ul was taken every hour and put in a 96-well plate. Absorbance at 595nm of each sample was then measured by an Envision Multiplate Reader. In total, for each bacterial sample, 2 trials of triplicates were used throughout the 8 hour period.
Of all our parts, BBa_K2764001 had the strongest promoter strength, followed by BBa_K2764002 and then by BBa_K2764003. The control, had the same promoter strength as BBa_K2764001.Taken individually, the absorbance-time charateristics of each strains are as follows:
Figure 1. Growth curve of bacteria transformed with BBa_K2764001.
According to Figure 1., the growth rate of bacteria with BBa_K2764001 rises steadily throughout the 8-hour period and peaks at 0.9AU at T=8
Figure 2. Growth curve of bacteria transformed with BBa_K2764002
.
Similarly, from Figure 2., 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
Figure 3. Growth curve of bacteria transformed with BBa_K2764003
.
Meanwhile, from Figure 3., 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.
Simply comparing the final absorbance of these 3 graphs, it can be safely said that the host cells with BBa_K2764001 has the strongest growth, followed by BBa_K2764002 and then by BBa_K2764003.
Then, when the 3 growth curves are compared with the control’s growth, the graph is as follows:
Figure 4. Growth curve of bacteria transformed with BBa_K2764001, BBa_K2764002, BBa_K2764003
.
From the graph, it can be seen that the control’s growth rate is higher than all 3 parts up until T=3. After which, the control has a slower growth rate than bacteria with BBa_K2764001 but still maintains a higher growth rate than bacteria with BBa_K2764002 and BBa_K2764004 respectively. Another thing to note is that from T=0 to T=2, the cell growth of bacteria with BBa_K2764001 is similar to that of both bacteria containing BBa_K2764002 and BBa_K2764003.
The higher growth rate of the control compared with that of the 3 parts in the first half of the experiment would indeed support the hypothesis that AlkL is the independent variable that affects host cell growth. It can be further ascertained that AlkL is toxic to the host and inhibits growth to a certain extent as the control has a higher growth rate initially. However, it is interesting to note that bacteria with BBa_K2764001, which has the highest promoter strength, could be able to have the highest growth rate compared with the other parts, and can even surpass the growth of the control in the second half of the experiment. Supposedly, the more AlkL is expressed, the more toxic the overall effect to the host is. That said, bacteria with BBa_K2764001 should have the weakest growth and BBa_K2764003 the strongest. Contrary to expectations, that is not what we observed.
We hypothesize that bacteria with BBa_K2764001 instead has the highest growth rate because AlkL also imports long chain fatty acids from the LB medium it was seeded in. LB itself contains yeast extract, which is obtained from the cell content of yeast, will contain long chain fatty acids. Similar to how FadL, a native E. coli fatty acid transporter, can sometimes mistakenly import medium chain alkanes into the cell, perhaps the AlkL in our transformed bacteria can also mistakenly import long chains of fatty acids, which can be β-oxidized in the cell’s native system as a nutrient. This will aid in the growth of the bacteria instead. As such, it may be that while certain amounts of AlkL produced by the cell inhibits cell growth, larger amounts of fatty acids are still imported into the cell because of it, which will boost cell growth. It is possible that in this push-and-pull scenario, the effect of boosting cell growth is more favored than the inhibition when more fatty acids are imported and this enables net cell growth to be slowly achieved over time. This is supported by the fact that the growth rate of bacteria with BBa_K2764001 is initially lower than the growth of control before rising slowly to overtake it, showing that the cell growth is slowly favored over time.
For the growth rate of BBa_K2764003 bacteria, there is relatively less AlkL but it seems it is already enough to inhibit growth to a large extent. While there is less AlkL, there is also less fatty acid transported, so perhaps in this push-and-pull scenario, inhibition of cell growth is favored over boosting of cell growth as not enough fatty acids are imported into the cell to offset the toxic effect of AlkL. It may be that from T=0 to T=1, the AlkL had not been expressed enough to reach toxic levels, so the growth of bacteria with BBa_K2764003 is similar to the rest. Then, after T=2, the amount of AlkL is enough to give a toxic effect, such that the effect of cell growth inhibition is favored over the effect of cell growth boost, hence, there is negative growth observed for bacteria with BBa_K2764003.
Given the extremes of cell growth inhibition and cell growth boost exhibited by bacteria with BBa_K2764003 and bacteria with BBa_K2764001 respectively, it is not unreasonable for bacteria with BBa_K2764002, which has medium promoter strength, to have cell growth between the other two. This also supports the push-and-pull hypothesis that suggests bacteria with BBa_K2764002 is able to offset some of the toxic effect of AlkL by importing fatty acids, but to a much lesser extent than bacteria with BBa_K2764001, resulting in stunted growth compared with the control. However, near T=8, it can be seen that growth of bacteria with BBa_K2764002 is almost the same of that of the control. This further supports the hypothesis that cell growth is slowly favored as more fatty acids are imported by AlkL over time into the cell to boost growth.
In conclusion, it can be said that AlkL certainly has an effect on host cell growth. It is proven to exhibit toxicity by its inhibition of host cell growth, as shown by the first half of the graph. However, it may also aid the growth of the host cell, as shown by the second half of the graph.
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].