Team:OUC-China/proof2
Proof of concept
Adhesion platform
We use ice-nuclei to display the monomer streptavidin on the surface of E.coli outer membrane and Aga1p-Aga2p system to display the biotin on the yeast cell wall. The two form a covalent linkage and bind with other firmly, establishing a co-express platform.
After sub cloning our target gene onto the vectors successfully, we introduced them to ei-ther E.coli or S.cerevisiae to confirm the expression and correct function of corresponding protein, which means they need to be integrated in the out membrane or cell wall. Therefore, we conduct Western Blot so that we can prove our result specifically. Af-ter that, we will need to verify that the heterogeneous cells do adhere to each other through the combination of biotin and mSA. With rhodamine-polyethylene glycol-biotin and FITC-streptavidin, we conducted immunofluorescence staining on both E.coli and S.cerevisiae, and by measuring two florescent, we had a visual validation of parts work. Meanwhile, we explored the optimal condition for their co-culture and drew a con-clusion after preliminary experiment that we will inoculate the yeast in the medium contain-ing galactose and introduce E.coli after 30 hours’cultivation.
For more solid a proof of their adhesion, we use Immunofluorescent localization to demonstrate their relative location and took photos with STM for their microstructure. What’s more, we would to acquired some quantitative data such as the percentage of link-age and expression with FCM. As a result, we can say that our co-expression platform was established successfully.
E.coli strain construction
We assume expressing monomer streptavidin on the outer membrane of E.coli will help us using biotin-avidin system to link E.coli and S.cerevisiae. In part registry, we find BBa_K523013 constructed by team Edinburg encoding INP-eYFP. We acquire INP using PCR from this part. Then we choose promoter J23106 from part registry because it can express protein continuously so that we can have a relative steady co-culture system. The mSA part is from team Peking that give us much help. Monomer streptavidin is much smaller than wild type and is easier to use in circuit. Based on above reasons, we designed J23106-INP-mSA-pSB1C3 plasmid and trans-form into DH5α strain.
Fig 2.1 Electrophoresis result of expression vector. Lane 3&4 is pSB1C3-J23106-INP-mSA.
After the sequencing results confirmed that we successfully cloned the J23106-INP-mSA-PSB1C3 expression vectors, we transformed the target plasmids to DH5α for amplification.
Protein expression verification
If the protein is successfully expressed, they will be displayed on the extracellular mem-brane of the E.coli. Therefore, we can verify protein expression by detecting whether INP-mSA is displayed in the outer membrane protein.
We used the Fractionation Separation to extract three components of the outer mem-brane protein, the intimal protein and the cytoplasmic protein of engineered E.coli. Further Western Blot aim to confirm the previous result specifically and We choose to use horseradish peroxidase-biotin because it have the specific ability to bind INP-mSA.
Fig 2.2 Western blot analysis using HRP-biotin to evaluate JIM expression. lane1, the outer mem-brane of JIM; lane2, the intimal protein of JIM; the cytoplasmic protein of lane3; lane4, the outer membrane DH5α; lane5, the intimal protein of DH5α; lane6, the cytoplasmic protein of DH5α.
Western Blot result shows that there is a clear protein band at corresponding position with a correct size and the result specifically. It makes sure that we do express INP-mSA on the E.coli surface successfully .
Protein function verification
After verifying the protein expression, we want to prove that the protein can function as normal. We used Rhodamine-biotin to immunofluorescence stain the engineerd E.coli and negative strains DH5α to observe whether the bacteria had fluorescence under fluorescence microscope.
The result proves that the function of protein was normal.
Fig 2.3 Rhodamine-biotin immunofluorescence staining of engineerd E.coli(left) and negative strains(right).
Meanwhile, we would like to quantify the expression of INP-mSA compared with DH5α with plate reader. When we carried out the experiment, we met the problem that there are different incubating conditions in different paper. So we had to explore the best incubating condition of the INP-mSA protein expression. We did two sets of experiments to compare the fluorescence values of 2h incubation under two conditions (0 ℃ and 30 ℃).
Fig 2.4 Expression level of INP-mSA fusion protein under different conditions.( I ) the fluores-cence values of E.coli DH5α cell expressing JIM fusion and DH5α under 0 ℃ incu-bation for 2h. ( II ) the fluorescence values of E.coli DH5α cell expressing JIM fusion and DH5α under 30 ℃.
The image showed that E. coli containing Jim Plasmid successfully expressed INP-MSA fusion protein. The fluorescence value was higher after incubation of dye 2h under 30 ℃ conditions. We speculate that there may be an ice dye incubation that causes ice nucleation proteins to form crystals that inhibit the combination of fluorescent dyes and the MSA of fusion proteins.
Moreover, we want to confirm the connection between E.coli and S.cerevisiae by FACS analysis, because this analysis method can observe in single-cell level. We can use Rhoda-mine-polyethylene glycol-biotin to dye the E.coli containing JIM Plasmid for char-acterizing the expression of the protein by fluorescence detection.
Unfortunately, however, we found the Beckman flow cytometry (FCM) in our university couldn’t excite our fluorescent dye rhodamine-biotin so we was not able to use it to catch dyed E.coli. In spite of this, luckily we found that we can use FCM to confirm the ex-pression of biotin and its expressing percent. We use FITC-streptavidin to stain on S.cerevisiae and use 488nm exciting light to excite it while receive it at 525nm. As the result showed, the fluorescent peak shifted towards the direction of stronger fluorescent, which means positive sample express more BAP. Compared with the results of empty sam-ple, we can ensure that BAP is displayed on the surface of Saccharomyces cerevisiae. Above the threshold, there are 48% of Saccharomyces cerevisiae in the positive sample. While in the negative sample and empty sample, the percentage is only 28%.
Fig 2.5
Yeast strain construction
Here we need to construct the vector of BirA (biotin ligase) and BAP (biotin acceptor peptide) in yeast for displaying biotin on the surface.
We first cloned birA into the yeast-shuttle plasmid pYC230. After transformation, it ena-bles the yeast to transfer biotin to biotin-receiving peptide bap. pYD1 is another 5.0 KB ex-pression vector designed for expression, secretion, and display of proteins on the extracel-lular surface of S.cerevisiae. The vector contains AGA2 gene from S.cerevisiae encoding one subunit of the a-agglutinin receptor. Fusing our gene of interest to AGA2, so we can enable the yeast to display the biotin on the extracelluar surface.
Fig 2.6 Electrophoresis result of expression vector. Lane1&2 is pYD1-BAP, lane 3&4 is pSB1C3-J23106-INP-mSA, lane5&6 is pYC230-BirA.
After successful construction of plasmids, we introduced pYD1 containing BAP and pYC230 containing Bira into S.cerevisiae EBY100.
Protein expression verification
We want to test whether biotinylated bap is successfully integrated in the yeast cell wall. So we dye engineered yeast and negative control respectively with FITC-streptavidin. Using fluorescence microscopy we are able to observe the fluorescence of both.
Fig 2.7 The fluorescent staining of yeast with FITC-Streptomyces. (left) S.cerevisiae EBY100 without target gene; (middle) S.cerevisiae EBY100 with BAP+Bira though galactose induction for 12h; （right) S.cerevisiae EBY100 with BAP+Bira though galactose induction for 32h.
Meanwhile, we would like to quanty the expression of biotin compared with EBY100 with plate reader and wonder when biotin can reach the maximum expression.
Fig 2.8 Expression level of biotin in different time compared with negative controll.
And after Xh, the expression level of biotin achieved the highest amount.
Co-cultivation verification
We aimed to build an adhesion platform of E. coli and S.cerevisiae. Therefore, first we need to select a culture condition under which the heterogeneous cells can both grow happily. Taking into account the expression of the BAP in S.cerevisiae, galactose is required to induce protein expression. And to avoid diauxic growth of yeast when changing a new carbon source in the medium, we ultimaytely selected the YNB-CAA galactose medium. In order for the optimize expression of INP-MSA fusion protein, the in-ducing expression of BAP, and the growth rate of two heterogeneous cells, we took 28 ℃ as the culture temperature.
We characterized the growth curve of E. coli with JIM target gene and that of S.cerevisiae containing BirA & Bap under the mentioned culture condition. And the dry group wonder some parameters of the growth of E.coli and S.cerevisiae, as well as the consumption of galactose.
Fig 2.9 (left) The growth curve of E.coli with JIM; (right) the growth curve of S.cerevisiae containing BirA & Bap.
From the image we can tell, when the yeast and E.coli grows at 28 ℃, the culture time needs to be longer than 48h in order to make the yeast take a dominant proportion. We adjust the YNB-CAA galactose medium containing S.cerevisiae initial OD600 to 1.
Meanwhile, we used HLPC to measure the consumption of galactose.
Linkage validation
In order to illustrate that in practice, the co-culture of S.cerevisiae and E.coli can truly form the collaborating platform of heterologous cells, we will cultivate the two strains together by adding E.coli to the galactose-induced yeast. The co-cultured samples will then be dyed with rhodamine-biotin and FITC-streptavidin.
As is shown below, we can see that there are quite a few areas of overlapping red and green fluorescent in positive group where there are both S.cerevisiae and E.coli in the sample, indicating that they are very likely to have linked to each other. The negative control of S.cerevisiae and E.coli however, has either red or green fluorescent. And for the empty group, there is no fluorescent detected at all.
Fig 2.10 The co-cultured samples will then be dyed with rhodamine-biotin and FITC-streptavidin（tips: please maximize the intensity of your screen when looking at this picture）
Finally, we use the scanning tunneling microscope to shoot the microscopic structure of the connecting system and get a clearer picture of the connection structure. So far, we can say our adhesion platform is successfully established.
Fig 2.11 Observation of scanning tunneling microscope
Cell Fractionation
At first Cells cultured at 37℃ until cell density(OD600=0.6-0.8), after cultured at 25℃ for 24 h. Harvested cells were washed, and resuspended in PBS buffer containing 1 mM EDTA and lysozyme at 10 Ag/mL. After 2 hincubation, cell suspension was treated with an ultrasound sonication at 30 sec 2 cycles. To obtain total membrane fraction, whole cell lysate was pelleted by centrifugation at 20,000 rpm for 1 h using an ultracentrifuge (Optima LE-80K; Beckman, Fullerton, CA). The supernatant was regarded as soluble cytoplasmic fraction. For further outer membrane fractionation, the pellet (total membrane fraction) was resuspended with PBS buffer containing 0.01 mM MgCl2 and 2% Triton X-100 for solubilizing inner membrane and incubated at room temperature for 30 min, and then the outer membrane fraction was repelleted by ultracentrifugation.Western Blot Analysis
An equal volume of each fraction (cytoplasmic and outer membrane) of the cells containing inp-mSA were mixed with SDS sample buffer (TaKaRa, 4X Protein SDS PAGE Loading Buffer), boiled for 15 min, and resolved by 12.5% (wt/vol) SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoretic transfer to Hybond-PVDF membranes with transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% methanol, pH 9.2) by using a Trans-Blot SD Cell (Bio-Rad, Hercules, CA) at 100V for 1h30 min. After blocking for 1 h in TBS buffer (20 mM TrisHCl, 500 mM NaCl, pH 7.5) containing 5% (wt/vol) nonfat dry milk, the membrane was then incubated for 1.5 h at room temperature in antibody solution (1% (wt/vol) nonfat dry milk in TTBS (TBS with 0.05% Tween-20)) containing HRP conjugated Biotin (1:500 vol/vol) (Sangon Biotech). After successive washing twice for 5 minutes with TTBS for 2 and first for 5 minute with TBS. Color, putting the membrane protein on the face on the petri dish, and then to the above color AB drops, reaction of 2-3 minutes, then don't directly put the cassette in the dry, covered with layers of plastic wrap, draw out the film edge, so as to avoid dark room inside can't find the location of the membrane.I use a petri dish to fill the film, then rinse it out and put it in the fixer.
Pyd1 yeast display vector expression and display of protein fusion
Grow untransformed EBY100, EBY100/pYD1, and EBY100/pYD1 containing your gene of interest as follows.1. Inoculate a single yeast colony into 10 ml YNB-CAA containing 2% glucose and grow overnight at 30°C with shaking.
2. Read the absorbance of the cell culture at 600 nm. The OD600 should be between 2 and 5. If the OD600 is below 2 or over 5, refer to the table below.
If the OD600 is....
Then you may...
below 2,
either continue growing the cells until the OD600 reaches 2 or proceed to Step 3.
over 5,
proceed to Step 3.
Note: In general, OD600 readings less than 2 will decrease the number of cells displaying fusion proteins. OD600 readings greater than 5 will delay induction of expression of the displayed protein.
3. Centrifuge the cell culture at 3000-5000 x g for 5-10 minutes at room temperature.
4. Resuspend the cell pellet in YNB-CAA medium containing 2% galactose to an OD600 of 0.5 to 1. This is to ensure that the cells continue to grow in log-phase. For example, if the OD600 is 2 from Step 2, resuspend the cells in 20 to 40 ml of medium.
5. Immediately remove a volume of cells equivalent to 2 OD600 units. For an OD600 of 0.5, remove 4 ml and place on ice. This is your zero time point.
6. Incubate the cell culture at 20-25°C with shaking.
Note: In general, more cells will display the protein fusion at 20°C. However, if you do not have an incubator that maintains 20°C, try 25°C.
7. Assay the cell culture over a 48-hour time period (i.e. 0, 12, 24, 36, 48 hours) to determine the optimal induction time for maximum display. For each time point, read the OD600 and remove a volume of cells that is equivalent to 2 OD600 units (see Step 5, above). Proceed to Staining of Displayed Proteins, below.
Staining of Displayed Proteins
For each time point, assay untransformed EBY100, EBY100/pYD1, and EBY100/pYD1 containing your gene of interest. Time points may be processed as they are collected or placed on ice and stored at +4°C until all time points are collected. Do not freeze cells.1. Take your time points from Steps 4 and 6, above, and centrifuge at 3000-5000 x g for 5-10 minutes at +4°C.
2. Resuspend the cells in 1X PBS and centrifuge as in Step 1.
3. Remove the PBS and resuspend the cell pellet in 250 µl of 1X PBS, 1 mg/ml BSA, and FITC-Streptavidin (1:500 vol/vol) (Solarbio) .
4. Incubate on ice for 30 minutes with occasional mixing.
5. Centrifuge the cells at 3000-5000 x g for 5-10 minutes at +4°C.
6. Wash the cells thrice with 1X PBS.
7. Resuspend the cells in 700μ 1X PBS to divided into 96 plates, and the fluorescence values were measured by the microplate reader, or to sample, biological section, and observed with a fluorescence microscope.
Co-culture
1.Yeast cell was cultured with YPD medium for 48 h at 30◦C.2.Then the yeast cells was transferred into YNB-CAA Gla medium as adjusted OD=1 by YNB-CAA Gla medium and cultured for 30h at 25 ◦C.
3.Then add E.coli into YNB-CAA Gla medium with OD=0.4，cultured for 12 h at 28 ◦C.
4.Then the cells can be used in observation by microscopy or FACS method.
FACS
1.After precultivation with appropriate selective medium for 18 h at 30◦C, yeast cells were harvested and washed with phosphate buffered saline (PBS) three times.2.The cell density was adjusted to OD600 = 1 by PBS.
3.Then cells were incubated with 20 g/ml of streptavidin–FITC (Solarbio)for 1 h.
4.After washing by PBS three times, cells were observed with FACS analysis.(Beckman)
5.FACS use as ‘Cytomics FC500 MCL with CXP software Instructions For Use’
Microscopy
1.After precultivation with YNB-CAA Gla medium for 12 h at 28◦C, yeast cells and E.coli were harvested and washed with phosphate buffered saline (PBS) three times.2.The cell density was adjusted to OD600 = 1 by PBS.
3.Then cells were incubated with 20 g/ml of streptavidin–FITC (Solarbio)for 1 h.
4.Then the cells were washed with phosphate buffered saline (PBS) three times.
5.Then cells were incubated with 20 g/ml of Biotin-Rhodamine (ToYongBio)for 1 h.
6.After washing by PBS three times, cells were observed with microscopy.
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