Eukaryote
Protein expression
Phicia pastoris is typical Eukaryotic expression host, but expressing plant genes is still a big challenge. So we firstly test whether our three interest genes can be efficiently expressed. We measured the Exogenous expression of PP2CA, ABF2 and SnRK2.2, We found there is no significant difference between the three proteins and it is worth noting that PP2CA level is less than the other two. But considering that PP2CA is a phosphorylase, the typical enzymatic reaction rate is fast enough to reduce the effect brought by the amount deficiency.
Fig1
Fig2
And then, we ran a SDS-PAGE to identify the three proteins(Fig2). The left figure shows that our target bands are shifted to the length between 72kD and 95kD. We supposed that the protein had been glycosylated
Fig3
Fig4
Bi-stable function:
We constructed expression plasmid and submitted this part (BBa_K2036030) to the registry. But on account of long-term , we didn’t have enough time to test its function. But our modeling simulated the switch functions. See to Eukaryote circuit modeling
Prokaryote
Protein&protein reaction
--CIII and Ftsh
We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014 ) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015 ) These two parts were to test the whether CIII can protect CII from being degraded by Ftsh by competitive inhibition. But we met problems transfer the segment from pSB1C3 to pET-Duet-1 expression plasmid. So we were late to put the result on wiki, but we would show it at the Jamboree.
Protein&promoter
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--CII and pRE
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--CI and pR
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--Cro and pRM
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CII (BBa_K2036000) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.
According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.And we also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.
CI is a repressor from bacteriophage lambda. And to test its interaction with pR promoter, we constructed CI-TT-pR-RBS-GFPLVAssrAtag-PET-Duet-1 and take pR-RBS-GFPLVAssrAtag-PET-Duet-1 as control to test its inhibition function.As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR to some degree but the leakage expression under pR can not be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit.
We also detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification (seen from the figure below).
We characterized Cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains Cro expressed less GFP protein than control group over time. It proves that Cro can effectively bind pRM to block its downstream gene’s transcription.
Tri-stable function
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--Preliminary experiments of ptrp2
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--Preliminary experiments of LVAssrA-tag
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Ptrp2(BBa_K2036000) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.
According to the GFP expression curve, we choce 50μM final concentration to induce ptrp2
In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used placI from the Rgistery (BBa_J04500) to characterize the degradation tag LVAssrA.
We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.
Fig: LVAssrAtag degradation rate measurement under placI
Seen from the figure, we are sorry to find the serious placI expression leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes.
Application
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Beta-galactosidase activity:
more details
Owing to limitation of time, we didn’t test lactic balance function of our engineered strain in vitro. But we tried to characterize plac-induced beta-galactosidase activity to prove that half of our bi-stable switch works.
We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.
Previous Improvements
HUST-China 2015 put up a 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 didn’t stopped this year -- 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:
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 successfully 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.
Sand cementation function
Last year, because of time limit 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 result 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).
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/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.
