Team:HUST-China/Results
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1. Light Control System
Before the whole circuit were determined, there were two kinds of light control system for us to choose: the CRY2-CIB1 system and the PhyA-FHL system. By DDEs modeling simulation of both systems, we found that the CRY2-CIB1 system fits our circuit better.
According to reference [1], to regulate DNA transcription by light, we use this CRY2-CIB1 system based on a two-hybrid interaction, in which a light-mediated protein interaction brings together two halves (a binding domain and an activation domain) of a split transcription factor. From addgene, we received a plasmid pRMH120 that containing both Gal4BD-CRY2 and Gal4AD-CIB1 fusions on a p414TEF backbone. These two fusions are under the control of constitutive promoter PTEF1 and PADH1 respectively. Since promoter PGal1 and downstream gene β-galactosidase exists in yeast Y187 originally, we can validate the light-control system by testing the activity of β-galactosidase. Thus, we use Saccharomyces cerevisiae Y187 as chassis to test the light-control system. In the near future, we will construct this system into JMY1212 supporting vector JMP62, transform into Yarrowia lipolytca and test the system again.
Experiments were carried out three times with similar results to show. We observed that pRMH120 transformed cells incubated in white light (18W) gave distinguishable activation compared to pRMH120 transformed cells incubated in total darkness, which means that GalBD-CRY2 coupled well with GalAD-CIB1 for light-inducible protein expression. Considering AD and BD could bind randomly, the result that the strain with pRMH120 in darkness was also activated a bit than the control wildtype yeast is reasonable.
2. Supporting System
The verification for supporting system consists of two aspects: the cell surface display system and the function of a series of silica-tag proteins.
2.1 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 shows the result of our verification of cell surface display system.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. But this result still successfully demonstrated the cell surface display of our silica binding proteins.
2.2 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. 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.
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.
In addition, this functional test result instructed our further experiment of sand cementation test.
3. Flocculating System
3.1 SDS-PAGE with Mcfp3
Firstly, we tried to identify the flocculating protein MCFP3 that is secreted outside cells. We concentrated cell culture solution of test mcfp3-JMY1212 cells and control wildtype cells, and then separated proteins by SDS-PAGE. Figure 4 shows an obvious ~12kDa protein bands of Mcfp3 in test lane, which cannot be found in control lane. This result proves that the Y.lipolytca JMY1212 can produce and release the flocculating proteins into surrounding environment.
3.2 Verification of Mcfp3’s Flocculating Function
To verify mcfp3’s function,we did flocculating test on the object slide
We added the concentrated culture supernatant from mcfp-3 secretion yeast or wildtype control, after coomassie brilliant staining and washing by buffer，Flocculated proteins remained obviously on the slide of mcfp-3 secretion yeast sample. This result shows secreted mcfp-3 has strong flocculating ability and can stick on solid surface.
4. Test of Sands Cementation with Our Tested Cells
To test the cementation ability of our testee cells(Si-tag+Mcfp3), we conducted a laboratory test of sands cementation. As we can see in Figure 5A, 40 gram quartz sands mixed with testee 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 testee cells , the sands aggregated obviously, and we can even obtain an intact sand cylinder (Fig 5B). We further compared the treated sands under microscope, we can find that the quartz sand granules treated with testee cells aggregated together (Fig.5C) while the quartz sand granules treated with wildtype cells are still dispersed.
This results shows that our testee 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 testee cells 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.
Future Plan of Experiments
1.Circuit completion and verification
we have succeeded in constructing part of circuit as the figure 1 and figure 5 shows,what we need to do next is piecing Panb1 together with the surface display module (figure 2),and transform all these circuit parts into JMY1212.Also we need to retransform the light control system (figure 4) into JMY1212 (we have done the verification of light control system in Y187). Then we will test whole circuit’s function, measuring the secretion or expression levels under the work of light control system.
2.Verification for ROX1 and Panb1
To verify the ROX1 and Panb1, we will piece Panb1 together with GFP (figure 3), and transform it into JMY1212 which containing the ROX1 module which we have constructed (figure 1), and then we will measure the fluorescence levels of GFP to verify the function of ROX1/Panb1.
3.As we have finished the Ptrp-GFP’s construction, we need to induce the Trp promoter (improved by us) and measure the fluorescence levels of GFP to test the Ptrp’s function.
