Engineering success | iGEM HUST-China

Engineering success


We want to build an enzyme system that can produce pigments and degrade them according to our needs. For the same idea, we need the second type of enzyme system to produce short peptides for perm, and it can also degrade short peptides at any time. In order to achieve the goal, we designed multiple devices and four systems. In the stage of pathway construction, we tried a variety of methods, summed up the experience, learned from mistakes, and finally achieved some results. Our exploration and work this year has also provided some valuable experience for the follow-up teams.

Cycle1:Construct by the homologous recombination method

Several homologous fragments are designed to link multiple genes altogether at one time, using seamless cloning, to construct a system with complete function. Some success are gained during construction, like the whole pathway of FMO dimer, while significant difficulties occur as the same time. Lessons are learnt and settlement are designed, in order to overcome the present reality of getting much shorter product than expected, after comparing our onlu success to tons of failure.

Cycle2:Construct by enzyme digestion and ligation

Classic, reliable but lenthy double-enzyme digestion and reconnection is used to construct basic devices, along with systems with complete function. Eventually, good news is we have succeeded in building all of our basic devices, but bad news is we have no choice but to put off the construction of complete pathways after iGEM, due to the precious but limited time, which undoubtly will be continuously carried forward.

Cycle3:Extracellular expression portion

In order to achieve our purpose of perming and dyeing hair directly, we originally designed to express all enzymes and short peptides outside the cell. So after Electrotransforming the plasmid into yeast, we directly tried to detect our target protein in the supernatant. After centrifugation, we took the supernatant for SDS-PAGE detection. But after many tests, only part of the target protein was detected. After analysis, we suspected that the target protein could not be detected because the extracellular expression was low and unstable, or due to the mismatch between the signal peptide and the spatial structure of the protein. So we want to try express in the cell later.

Cycle4:Intracellular expression portion

After determining that under the existing system, the extracellular expression of some proteins may not be achieved. We chose to reconstruct the plasmid, remove the signal peptide and try to express it in the cell. So we amplified the target gene sequence without signal peptide by PCR and inserted it behind the Panb1 promoter to construct a plasmid without signal peptide. Also after electrotransformation, we extracted the total protein of yeast, purified it with nickel column and examined it by SDS-PAGE. This time, we successfully detected the rest of our protein and realized the synthesis of indigo and lycopene.

I think the methods in constructing engineering plasmids are quite valid, you? Well, I have a question here. What if you or I want to repeat their experiment? I want to know what protocols did they follow to get those result.


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