Difference between revisions of "Team:HUST-China/project/inprovement"

Overview

HUST-China 2015 put up an original method to cement sands as a promising way to help build firm structure in marine environment. The project “Euk.cement” was nominated “Best Environment Project” and “Best New Basic Part” in 2015.

Fig1: HUST-China 2015 Euk.cement circuit

Our engineered circuit (Fig1) is turned on in darkness just like the dark condition when our strain gets into the sands. The structural-reversed CRY2 will dissociate with CIB1 (the light sensitive part is based on yeast two-hybrid system). Without CRY2-BD’s target function, AD is hard to combine and activate pGal1 in the complex environment of nucleus, so the downstream ROX1 is no longer transcribed. With the degradation of translated ROX1 protein, pAnb1 starts to express the downstream gene as the supporting matter and flocculating agent :LIP2 pro-Si-tag-YLcwp3 and LIP2-pro-Mcfp-3 fusion proteins. The Si-tag will be displayed on the cell wall to help immobilize the strain on the sands and then the secreted viscous protein Mcfp-3 will function as glue to bind sands together. And the final step is the CaCO3 crystallization to enhance the cementation. (CO2 produced by cell’s respiration reacts with Calcium ions in marine environment )

After the Jamboree,HUST-China iGEMers stepped forward--We did more part characterizations and achieved more valid data to submit to the registry. What’s more exciting is that we successfully published a paper “A living eukaryotic auto-cementation kit from surface display of silica binding peptides on Yarrowia lipolytica”on ACS Synthetic Biology(IF 6.076).

Flocculation system

The circuit consists of the promoter anb1, LIP2 prepro signal peptide, Mcfp-3 flocculating protein and its terminator yADH1 (Fig2). Mcfp-3 is the main flocculating material. It can perform self-assembly and thus get high affinity to silica and other solid surface. So the secreted Mcfp-3 protein can crosslink sand particles together and complete flocculation.

Fig2: Flocculating circuit

Last year we ran a SDS-PAGE to identify the flocculating protein MCFP3. Moreover, we tested its function by applying the concentrated supernatant on an object slide and Microscopic observation after staining. The microscopy result qualitatively indicated the successful secretion of MCFP3.

This year, we made a further step-- BCA(bicinchoninic acid) quantification methods to describe the MCFP3 secretion level. We took wild Yarrowia lipolytica cultrue as control group to verify the proportion that MCFP3 accounted for in all secreted proteins.

Sand cementation function

Last year, because of the limit of time, we only tested sand cementation function of the ST123-JMY1212&mcfp3-JMY1212 mixed cells. It showed obvious effect on cementing sands.

To make the data valid, this year, we together characterized 8 combinations of Si-tag domains and MCFP3 producing cells, and the results were quite corresponding to our expectation.