Part:BBa_K5052330
Alcohol Dehydrogenase II (AdhB)
About
In the obligatory fermentative bacterium Zymomonas mobilis, the regeneration of NAD+ depends upon two enzymes, pyruvate decarboxylase and alcohol dehydrogenase II. AdhB or alcohol dehydrogenase II catalyzes the conversion of acetaldehyde into ethanol. Z. mobilis is the only known obligately fermentative prokaryote that utilizes an Entner-Doudoroff pathway for glycolysis and, like Saccharomyces cerevisiae, produces ethanol and carbon dioxide as dominant fermentation products. Pdc encodes pyruvate decarboxylase which catalyzes the decarboxylation of pyruvate to produce acetaldehyde. In turn, adhB encodes alcohol dehydrogenase II which catalyzes the final step of ethanol fermentation by reducing acetaldehyde to ethanol, using NADH as a cofactor. Introducing adhB and pdc genes in our cyanobacteria would allow it to produce ethanol as a fermentation product, and it would be secreted out of the cell into the environment. The use of adhB and pdc for the production of ethanol in cyanobacteria was inspired by the University of Uppsala iGEM 2009 Team 1 These parts have been codon optimized for PCC 6803, 7942, and 7002.
Testing Functionality
To characterize functionality of parts and see if the codon optimization had affected enzyme production in E. coli, we tested BBa_K5052930/BBa_K5052931 in E. coli BL21 and analyzed using Schiff’s reagent. Schiff’s reagent reacts with aldehydes, primary, and secondary alcohols to form a compound that appears as magenta. We used this property of Schiff’s reagent to a) test for the presence of ethanol and b) quantify the amount of ethanol produced when compared to a standard curve.
E. coli BL21 was transformed with BBa_K5052930/BBa_K5052931 and grown at 37 °C in LB-kan in the dark. Transformants were also plated onto LB-agar-kan plates and left to grow at 37 °C overnight in the dark. The next day, ethanol was extracted via hydrogel distillation. Liquid cultures were pelleted and supernatant was extracted. 1” x 1” Spenco 2nd Skin Squares hydrogels were soaked in supernatant. 1” x 1” Spenco 2nd Skin Squares hydrogels were laid over cultures grown on solid plates. Before distilling, hydrogels were removed from supernatant and from cultures grown on solid media and were heat treated at 80C to remove any potential cells still remaining on the hydrogel. Hydrogel was then placed on watch glass and covered with 50 mL beaker collection vessel and allowed to distill at 80C for 5 min. Distillate collected from the collection vessel was moved to 1.5 mL microcentrifuge tube. This process for collecting distillate was repeated several times until 100uL of distillate was collected.
This distillate was then tested for ethanol by adding Schiff’s reagent to 10% v/v. Tubes were agitated by inverting and allowed to sit for 1 minute. Observed color change was quantified on ImageJ and compared against a standard curve of 10% Schiff’s added to 10%, 20%, 30%, 50%, and 70% ethanol after incubation for 1 minute.
Distillation allows us to remove any trace components in the media and isolate water and ethanol. When Schiff’s reagent was added to 10% v/v, color change was observed in distillate from BL21 samples and not in LB distillate. This demonstrates the activity of BBa_K5052930/BBa_K5052931 in E. coli.
References
(1) Team:Uppsala-Sweden - 2009.igem.org. Igem.org. 2009.igem.org/Team:Uppsala-Sweden (accessed 2024-09-30).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 711
- 1000COMPATIBLE WITH RFC[1000]
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