Revision as of 08:23, 16 September 2015

LIP prepro + E. coli ribosomal protein L2 (1-273aa) + 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.

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. Though due to the low resolution of our fluorescent microscope camera, we cannot show much clearer photos, but this result still successfully demonstrated the cell surface display of our silica binding proteins.

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.

Test of sands cementation with Euk.cement cells

Figure 4: Sands cementation with Euk.Cement. A: Sands cementation test was carried out in lab with trial column and quartz sands. B, C: Sands treated with Euk.cement cells (Si-tag+Mcfp3) form cementation in columns. We can see sands were stacked together by microscopy. D, E: Sands treated with wildtype control cells cannot form cementation and magnification is 10x10.

To test the cementation ability of our Euk.cement cells, we conducted a laboratory test of sands cementation. As we can see in Fig A, 40 gram quartz sands mixed with Euk.cement cells or control wildtype cells were loaded into each glass column, while solution carrying oxygen, calcium and culture nutrient was supplied in tubes under the impulse from peristaltic pump.

After 24 hours treatment, we dehydrated our sands columns in drying oven, and then took out the sands from the column. We can see the sands treated with control wildtype Yarrowia lipolytica JMY1212 are still scattered, only a few small solids can be found, these may be induced by the respiratory action of cells. However, by the treatment of Euk.cement cells (Si-tag+Mcfp3), the sands aggregated obviously, and we can even obtain an intact sand cylinder (Fig B). We further compared the treated sands under microscope, we can find that the quartz sand granules treated with Euk.cement cells aggregated together (Fig C) while the quartz sand granules treated with wildtype cells are still dispersed.

This results shows that our Euk.cement cells actually works well to make the silica particles form certain intact structure. which fits our cementation function hypothesis and design. We can also find in the figure that there are some small holes in the sand cylinder. This special structure indicated the balance between CO2 released from cell respiration and calcium sedimentation caused by released CO2. This is the final and vital step of the Euk.cement cell cementation process. In some cementation utilization, this structure is very important. For example, in desert sands solidation treatment, the multiporous structure will eliminate the potential compaction risk that may reject plants to grow. In artificial reef construction of aquafarm, the multiporous structure will also offer harbors to all kinds of marine lifes.

Application of the part

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

Figure 5-1: 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 5-2: Simulation of Surface Display System of Si-tag

From figure 5-2, 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.

Sequence and Features