Team:HUST-China/project/inprovement

Interlab

Previous Improvement

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.

Fig 1: HUST-China 2015 Euk.cement circuit

Our engineered circuit (Fig 1) 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 )

More information see to HUST-China 2015

After the Jamboree, HUST-China iGEMers stepped forward--We did more part characterizations and achieved more solid 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

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. It was noteworthy that BCA quantification of the total protein in concentrated cell culture suspensions showed that the engineered cells released a protein level 0.1 µg/µL higher than that of the control group.

Fig 2: Flocculating circuit

Fig3：BCA assay

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Flouculation
Part number	Description	Function
BBa_K1592000	LIP2 prepro(signal peptide)	As a signal peptide to secrete Mcfp-3 and Si-tag out of the membrane
BBa_K1592001	Mytilus californianus foot protein 3(Mcfp3) variant 3	Vicious protein to help bind sands together.
BBa_K1592003	Mcfp3 with LIP2 prepro	Fusion protein to be secreted out of the membrane to bind sands together

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 solid, this year, we characterized all 8 combinations of Si-tag domains and MCFP3 producing cells, and the results were quite consistent to our expectation.

Figure 4: Sand cementation test with Si-tag and MCFP3 producing cells. (a) Test facility for sand cementation in lab with trial column and quartz sand. (b) Sand treated with Si-tag and MCFP3 producing cells formed cementation in the column. (c) whereas sand treated with wild type control JMY1212 cells did not form cementation. (d) Sand particles from the Si-tag and MCFP3 producing cell treated column were evaluated using microscopy and were found to be stuck together. (e) but sand particles from the wild type control JMY1212 cell treated column did not stick together. (f) Microscopy image of sand treated with Euk.cement cells in flasks on a shaker, which mimics the real conditions of high water-to-sand ratio and turbulence-like waves. (g) Sand treated with control cells in flasks on a shaker. (h) Sand treated with Euk.cement cells in column forms a cemented cylinder. (i) Standardized 1cm 3 cube was modified from cementation sand cylinder and put on a platform weight scale. Weight was added onto the cube and the critical pressure value at cube destruction was recorded, and then normalized by the highest value. Quantification showing the different intensity of cylinders from the cementation of sand treated with different cells (quantification: n=3, t-test *: P&lt0.05).

The cementation test verified the cooperation of immobilization system and flocculation system cells in actual application conditions. Quartz sand (40 grams) mixed with either Si-tag+MCFP3 YMY1212 cells or control wild-type cells was loaded into a glass column, and a solution carrying oxygen, calcium and culture nutrients was supplied into the column using a peristaltic pump. After 24 hours of treatment, the sand columns were dehydrated in a drying oven and then removed from the glass column. The sand treated with control wild-type JMY1212 cells was still scattered; only a few small clumps could be identified, and these may have been induced by the constitutive respiration of the wild type cells. However, with the treatment of Si-tag+MCFP3 cells, the sand aggregated, and an intact solid sand cylinder was obtained. With further comparison of the treated sand under a microscope, the quartz sand granules treated with Si-tag+MCFP3 cells were found to be tightly agglutinated, whereas the quartz sand granules treated with wild type cells remained dispersed.

This result indicated that Si-tag+MCFP3 cells actually worked well at making silica particles form a certain intact structure, which fits our hypothesis and design of their cementation function. It was also noticed in the cementation test that there were some small holes in the cemented sand cylinder. This special porous structure indicated the balance between the CO2 released from cell respiration and the calcium or magnesium sedimentation caused by the released CO2. This sedimentation, however, will be the final and vital step of the cementation process. Indeed, in some cementation applications, this structure is very important. For example, in desert sand consolidation treatment, this multi-porous structure will eliminate the potential compaction risk and will enable organisms to grow on it; in artificial reef construction on aqua farms, the multi-porous structure could also offer niches to all types of marine life.

To mimic the real conditions in underwater applications, we also tested the sand cementation under the condition of high water-to-sand ratio with turbulence in flasks on a shaker. Compared to sand treated with wild-type cells, sand treated with Si-tag cells and MCFP3 cells was found to be cemented together tightly using microscopy.

To find whether sand treated with different Si-tag cells can form cementation with different intensity, column cementation tests were also conducted with MCFP3 cells and different Si-tag cells. Sand treated with all Si-tag cells except wild-type control cells could form a cemented cylinder. The relative intensity of the cylinders was quantified by the critical pressure value at cementation destruction. The results showed that Si-tag1+2+3 provided the highest cementation intensity, whereas Si-tag2+3 provided medium cementation intensity and the other strains provided weak cementation intensity. This finding is comparable to the results from the Si-tag silica binding test in which Si-tag1+2+3 cells provided the highest silica binding intensity while other cells provided medium or weak intensity.

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