Project Description

Overview

This year, our team is working on biofuel production using E. coli. In the US, most biofuel production comes from either corn or sugar cane to produce ethanol. Both of these crops require arable land and pull resources from the food supply. Algae is another option, but it requires land area for growing ponds. E. coli, on the other hand, can be grown in fermenters in a factory and do not affect the food supply or remove arable land from other productive use. E. coli naturally produces several alcohols that can be used as biofuels, including isopropanol, isobutanol, ethanol, and sec-butanol. Unfortunately, E. coli also has pathways that break down these alcohols when the concentration increases to prevent toxic levels from occurring. The combination of increasing the natural resistance to these alcohols as well as up-regulating the production of one or more of these alcohols could make this production methods commercially viable.

E. coli

Escherichia coli (E. coli) bacteria normally live in the intestines of people and animals. Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract. The types of E. coli that can cause diarrhea can be transmitted through contaminated water or food, or through contact with animals or persons.

E. coli consists of a diverse group of bacteria. Pathogenic E. coli strains are categorized into pathotypes. Six pathotypes are associated with diarrhea and collectively are referred to as diarrheagenic E. coli.

Shiga toxin-producing E. coli (STEC)—STEC may also be referred to as Verocytotoxin-producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC). This pathotype is the one most commonly heard about in the news in association with foodborne outbreaks.
Enterotoxigenic E. coli (ETEC)
Enteropathogenic E. coli (EPEC)
Enteroaggregative E. coli (EAEC)
Enteroinvasive E. coli (EIEC)
Diffusely adherent E. coli (DAEC)

Results

Figure 1 shows the survival percentage of E. coli under different concentrations of Ethanol.

Figure 2 shows the survival percentage of E. coli under different concentrations of Isobutanol.

Figure 3 shows the survival percentage of E. coli under different concentrations of Isopropanol.

Figure 4 shows the survival percentage of E. coli under different concentrations of 2-butanol.

Figure 5 shows that the plasmid carrying GlmY gives a small but significant increase in the resistance to low concentrations of isobutanol even though no promoter is present. At 60 ul isobutanol, the control had 12% survival while the culture with the GlmY plasmid had 25% survival. This increased resistance to isobutanol did not persist at higher concentrations.

Conclusion

In the graph, the X-axis represents the amount of Isobutanol in the alcohol. The Y-axis shows the survival percentage of E. coli when there is Isobutanol present. The circles represent the control culture, while squares represent the culture with the GlmY plasmid. Our team performed numerous sets of kill curve and recorded OD600 of tubes containing the different concentration of Isobutanol. The beginning of both curves is at (0, 100), showing 100% survival rate of E. coli without Isobutanol. One of the curves is the line of Control. The curve of control indicates how E. coli resists in Isobutanol when there is no genetic engineering added. The other curve is made after we put gene GlmY into E.coli plasmid. We can compare two curves and discover that the curve of GlmY is above the control curve, meaning that the survival rate of E. coli is higher with GlmY.

Future Development

The team has successfully amplified construct BBa_K2221002. For future work, we will run numerous sets of E. coli kill curve with construct BBa_K2221002 and plot a baseline-corrected graph of GlmY+J23102 promoter. Also, the team will look at increasing E. coli resistance against other alcohols, including 2-butanol and Ethanol, incorporating different constitutive promoteres, and increasing efficiency of alcohol production using adhE, adhF, Ipd genes for ethanol production, pta genes for acetate production, and AlsS, llvC, llvD, and Kdc genes for isobutanol production.

Figure 1 shows isobutanol production pathway E. coliFigure 2 shows ethanol and acetate production pathway by E. coli

References

General Information. (2015, November 06). Retrieved October 20, 2017, from https://www.cdc.gov/ecoli/general/index.html
Atsumi, S., Wu, TY., Eckl, EM. et al. Appl Microbiol Biotechnol (2010) 85: 651. https://doi.org/10.1007/s00253-009-2085-6
Munjal, N., Mattam, A. J., Pramanik, D., Srivastava, P. S., & Yazdani, S. (2012). Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli.Microbial Cell Factories,11(1), 145. doi:10.1186/1475-2859-11-145