Part:BBa_K1592013

LIP prepro + E. coli ribosomal protein L2 (1-60,GS linker,202-273aa) + YLcwp3

This is the cell display system of Yarrowia lipolytica, composed of LIP prepro, interest protein, and YLcwp3. LIP prepro is signal peptide used to secrete the interest protein out of the cell, and the YLcwp3 is the anchor domain binding the interest protein to the cell wall of yeast. We use this system to display silica-tag and test its binding characteristics.

E.coli ribosomal protein L2 was found to bind tightly to silicon particles, which have surfaces that are oxidized to silica. This L2 silica-binding tag, called the "Si-tag," can be used for one-step targeting of functional proteins on silica surfaces. The silica-binding domains of E. coli L2 was mapped to amino acids 1–60, 61-202 and 203–273, called Si-tag1, Si-tag2, Si-tag3. We respectively test the silica-binding characteristics of this three regions and their combinations.

This part is to diplay the amino acids 1-60,GS linker and amino acids 203-273 of E.coli ribosomal protein L2, Si-tag1+GS linker+3.

Usage and Biology

Here we use this system to display Silica-tag on cell wall of Yarrowia lipolytica to binding the silica, as the figure 1-2 shows.

E.coli ribosomal protein L2 was found to bind tightly to silicon particles, which have surfaces that are oxidized to silica. This L2 silica-binding tag, called the 'Si-tag', can be used for one-step targeting of functional proteins on silica surfaces.

Figure 1-1: The cell wall display system of Yarrowia lipolytica to display Si-tag.

Figure 1-2: The preview of Si-tag displayed on cell wall and work.

Characterization

Functional verification of cell surface display system

We used the fluorescence immunoassay to verify the success of cell surface display system. We had added the DNA sequence of 6xhis tag between the signal peptide and our silica-tag protein when constructing JMP62 plasmid, so that the 6xhis tag could be fusion expressed with the silica-tag protein and displayed on cell surface together. While the signal peptide could be cut out during the secretion. When we used the fluorescence immunoassay anti 6xhis tag, the primary antibody (mouse anti 6xhis tag) and the secondary antibody (FITC tagged goat anti-mouse IgG) detected 6xHis tagged Si-tag protein on cell surface.

Figure 2: Surface green fluorescence from anti si-tag-6xhis immunoassay was observed under 40X objective lens（Control is wildtype Yarrowia lipolytica JMY1212 without plasmid. Test cell is the JMY1212 transformed with JMP62 plasmid. Regional enlargement shows a surface display of FITC labled Si-tag-6xhis protein）

Figure 2 shows the result of our verification of cell surface display system. The fluorescence surrounding cell wall shows that we succeed in displaying the silica-tag protein onto the cell surface. For the limit of experimental conditions, we can not get a thorough fluorescence staining. Some cells can show a considerable flourescencent intensity, while some performs partial or weak flourescence which can not be detected by our microscope camera.

Silica surface binding test

After proving the success of the cell surface display system (which means our silica-tag protein displayed on the cell surface), we did the function test of silica binding proteins. To achieve different binding intensity for different cementation utilization, we constructed a series of silica-tag proteins containing different structural truncations under the control of promoter hp4d(BBa_K1592004). And we tested their different combining effects with silica.

As figure 3 shows, there are eight testing groups in total, these testing groups are named si-tag1, si-tag2, si-tag3, si-tag1+2, si-tag1+3, si-tag2+3, si-tag1+GSlinker+3, si-tag1+2+3 respectively according to the corresponding structural domain combinations. The cells was loaded onto glass slides, reserved for 10min and then wash with binding buffer for 3 times. The numbers of cells loaded before wash and reserved after wash was counted.

Figure 3: Silica binding test result of different surface displayed silica binding tags shows we achieved 3 different binding intensity for different cementation utilization. (Control is the wildtype JMY1212 without transformation)

As we can see from figure 3, all the test groups show obvious silica binding effects than the control under the same expression situation. We achieved 3 different silica binding intensity , the Si-tag3, Si-tag1+2, Si-tag1+3 strains show weak binding intensity, the Si-tag1, Si-tag2, Si-tag2+3, Si-tag1+GSlinker+3 strains show moderate binding intensity, while the Si-tag1+2+3 strain, which contains the full length of silica binding protein, have stronger silica binding intensity. It means that each protein structural domain has different silica binding ability. So we can choose different combinations of Si-tag domains to satisfy our different requirement of binding intensity in different cementation utilization.

Application of the part

the cell curface display system of Si-tag after darkness induction system.

Figure 4-1: The circuit of Darkness induction system and cell surface display system of Si-tag.

Modelling

With the DDEs model we built, we could run the simulation of the expression of Si-tag and determine its amount at any time. To test the function of our darkness induction system, the timeline would be set as darkness-light-darkness.

Figure 4-2: Simulation of Surface Display System of Si-tag

From figure 4-2, we can see that the innerSi-tag remains at a low concentration and the outerSi-tag accumulates very efficiently when engineered yeast is in darkness for the first time (0-200min). However, when exposed to light (200-500min), the expression of Si-tag is blocked and the rate of outerSi-tag accumulation decreases greatly. After light exposure (500-1500min), the expression of Si-tag and the rate of outerSi-tag accumulation gradually recover. Generally speaking, the darkness induction system is capable of controlling the downstream system and the expression of Si-tag is sufficient.

Improvement

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”. 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). We will demonstrate our working details in the following:

Sand cementation function

Last year, because of the limited 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. 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 (Figure 4a). 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 (Figure 4 c), 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 (Figure 4b). 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 (Figure 4 d, e).

These results 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/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.

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 1cm3 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<0.05)

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 (Figure 4f, g). 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 (Figure 4h). 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 (Figure 4i). 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 100
  Illegal XhoI site found at 605
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 626