Our Contribution

Overview

Our team aims to produce a biofuel in an environmentally friendly and economic way. We genetically engineered Clostridium tyrobutyricum (C.tyrobutyricum), reconstructing its metabolic pathways and allowing it to express exogenic proteins. The modified C.tyrobutyricum can synthesize butyl butyrate, a novel and well-performing biofuel, from shrimp shells, a common yet often wasted kitchen residue rich in nitrogen.

We have reached the following achievements.

1. Characterization of the part BBa_K1033910 (fwyellow)

Our team obtained the fwyellow gene from iGEM13_Uppsala and transformed E.coli strain BL21 with the plasmid vector pET-29a-fwyellow. We extracted and purified the fwyellow protein expressed by sonication, ultrafiltration, Ni-affinity chromatography, second ultrafiltration, and boiling. The fwyellow produced was analyzed by bio-information analysis (by SWISS-MODEL), electrophoresis, mass spectrum, and chromatography.

To Part

2. Hardware: Crushing and drying machine

Our hardware team designed a crushing and drying machine for cleaning and decomposing shrimp shells for this project. Grinders on the market have no drying effect, which can’t meet the needs of our criteria of raw material substrate. The design of this crushing and drying machine can improve the crushing efficiency of shrimp shells into dried powder suitable for the next step production needs. Future iGEM teams having demands for crushing hard materials into dried powder will find this machine very useful.

Figure 2. Model of our crushing and drying machine

To hardware

3. Hardware: Storage tank

We designed storage tanks for butyl butyrate. Current storage tanks often have rectangular observation windows with limited viewing angle and do not have a sun visor. We designed a circular window to expand the observation field and added a sun visor to the window to effectively avoid deterioration of the fuel by direct sunlight through the window. In addition, commonly used horizontal storage tanks occupy large space and many factories have limited warehouse. Our storage tanks have an angle adjustment design, so that they can be placed horizontally or vertically. Vertical position can be used when storage space is limited.

Figure 3. Model of our storage tank

To hardware

4. Plasmids constructed

We successfully constructed four plasmids pMTL-Pthl-adhE2, pMTL-Pthl-adhE2-thl-hbd, pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd, and pet25b-T7-pelB-CALB-ChBD. Detailed information of the proteins expressed in these plasmids and their functions are listed below. Future iGEM teams that need to use these genes to realize the corresponding functions can utilize these plasmids.

Gene and corresponding protein explanations:

• - Thiolase (thl): an enzyme derived from C.tyrobutyricum. It catalyzes the transformation of acetyl CoA to acetoacetyl CoA, one speed-limiting reaction in C.tyrobutyricum’s butyrate synthesis metabolic pathway. Overexpressing this gene increases the yield of butyrate.
• - Hydroxybutyryl coenzyme (hbd): an enzyme derived from C.tyrobutyricum. It catalyzes the transformation of acetoacetyl CoA to 3-hydroxybutyryl-CoA, one speed-limiting reaction in C.tyrobutyricum’s butyrate synthesis metabolic pathway. Overexpressing this gene increases the yield of butyrate.
• - Alcohol dehydrogenase (ahdE2): an enzyme derived from C.acetobutylicum. It catalyzes the transformation of butyryl-CoA to butyraldehyde, and finally to butanol. Expressing this gene enables butanol synthesis.
• - CALB (Candida antarctica lipase B): an enzyme derived from C.antarctica. This is one of the most efficient and widely used lipases to catalyze the esterification reaction, in which butyrate and butanol are transformed into butyl butyrate. Expressing this gene enables butyl butyrate synthesis.