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  • Microtubule

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Results

Microtubule

Display module

Plasmid construction

We have accomplished the construction of two parts whose functions are described respectively in the previous design page (Microtubule module). They are pYD1-α tubulin (BBa_K2220019) and pYD1-β tubulin (BBa_K2220020), both of which have been validated by sequencing. The electrophoresis image of these two parts are shown below (see Figure 1).
pYD1-β tubulin was transfected into S. cerevisiae EBY100 by our team and its function was verified by protein analysis techiniques including Western blot and immunofluorescence microscopy (see Figure 2 and 3). Meanwhile, pYD1-α tubulin was transfected into S.cerevisiae EBY100 by FAFU-China as a part of collaboration works.(Click to see more details)

Figure 1 The electrophoresis image of 6 plasmids.

Protein expression analysis- Western blot

Recombinant S.cerevisiae EBY100 strain harbouring the pYD1-β tubulin plasmid was precultivated to mid-log growth phase and then induced by galactose. After 24 hours of inducing，the supernatant from cell lysates of the engineered EBY100-pYD1-β was analysed by Western blot. The image shows the results of the Western blot analysis carried out with an anti-V5 antibody.

Figure 2 The partial results of the Western blot analysis carried out with an anti-V5 antibody.

Function analysis- Immunofluorescence microscopy

Recombinant S. cerevisiae EBY100 strain harbouring the pYD1–β tubulin plasmid was precultivated to mid-log growth phase and then induced for 24 hours at 20℃. During the inducing period, cells equivalent to 2 OD₆₀₀ units were collected every two hours from 8 h to 24 h. To detect the displayed protein, immunofluorescence microscopy was performed with mouse IgG against βI tubulin and donkey anti-mouse IgG conjugated with Cy3 as primary and second antibody respectively. Results showed that optimal detection of β-tubulin occurred at 12 h.

Figure 3 Induced 12h in SG-CAA medium；
A,B Recipient strain with empty plasmid;
C Bright-field micrograph of S. cerevisiae EBY100 cells harbouring pYD1–β tubulin;
D Immunofluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYD1–β tubulin.

Secretory module

Plasmid construction

Four parts have been constructed, which are pYCα-α tubulin (BBa_K2220022), pYCα-β tubulin (BBa_K2220023), pYCα-mCherry-α tubulin (BBa_K2220024), pYCα-β-tubulin-mGFP (BBa_K2220025) and pYCα-mCherry (BBa_K2220021). All parts have been validated by sequencing and electrophoresis.(See Figure 1)

Protein expression analysis- Western blot

Figure 4 An obvious color of mCherry produced by our engineered yeast harbouring vector pYCα-mCherry-α .

Recombinant S. cerevisiae INVSc1 strain harbouring pYCα-α tubulin, pYCα-β tubulin, pYCα-mCherry and pYCα-mCherry-α tubulin plasmid were precultivated to mid-log growth phase respectively and then induced for 48 hours at 30℃ in SG-Ura medium. The recombinant proteins were extracted from cell lysates and analysed by Western blot. The image shows the results of the Western blot analysis carried out with an anti-V5 antibody.

Figure 5 Western blot analysis of the supernatant from cell lysates of engineered yeasts mentioned above, carried out with an anti-V5 antibody.

Furthermore, it has been proven that our recombinant proteins can be secreted normally as expected. Firstly, the secretion function of part pYCα-mCherry have been proven by western blot analysis (See Figure 5). Then we tested the dynamic behavior of our recombinant proteins mCherry-α tubulin and β tubulin, which is described in the following functional analysis.

Figure 6 The results of Western blot analysis carried out with an anti-V5 antibody.
Lane A The purified supernatant of S.cerevisiae INVSc1 harboring pYCα-mCherry culture, induced for 12 hours in SG-Ura.
Lane B The supernatant from cell lysates of S.cerevisiae INVSc1 harboring pYCα-mCherry (without purified), induced for 12 hours in SG-Ura.

Protein expression analysis- Fluorescence microscopy

Recombinant S. cerevisiae INVSc1 strain harbouring the pYCα–mCherry-α tubulin or pYCα–mCherry plasmid was precultivated to mid-log growth phase respectively and then induced for 20 hours at 30℃. To detect the protein expression of our engineered yeast, fluorescence microscopy was performed. As the images below show, the expression rate of mCherry is almost up to 100%.

Figure 7 Induced 20h in SG-Ura medium；
A,B Recipient strain with empty plasmid;
C Bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry-α;
D Fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry-α;
E Bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;
F Fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry.

Recombinant S. cerevisiae INVSc1 strain harbouring pYCα–β tubulin-mGFP plasmid was precultivated to mid-log growth phase and then induced for 18 h at 30℃ in SG-Ura medium. The expression of recombinant protein can be obviously observed in the fluorescence microscope field.

Figure 8 Induced 18h in SG-Ura medium;
A,B Recipient strain with empty plasmid;
C Bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–β tubulin-mGFP;
D Fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–β tubulin-mGFP.

From the images above, we can conclude that all of our parts can work as expected, including display and secretion of recombinant proteins. Then, we tested the function of our upgraded display system.

Protein function analysis-　OD₃₄₀ test & Electron microscopy

Tubulin polymerization assay is based on an adaptation of the original method of Shelanski et al.(1973) and Lee et al.(1977). Light at a wavelength of 340 nm is scattered by microtubules proportionally to the concentration of polymerized microtubule. Purified α and β tubulins secreted by engineered INVSc1 were mixed together and incubated at 37℃ for 1 hour, and absorbance readings at 340 nm were conducted every minute. The results are shown in the image below.
Comparing the absorbance curves obtained, it was clear that the secreted tubulins had successfully polymerized into microtubules when GTP is added into the system.

Figure 9 Absorbance curve of polymerization reaction at 340 nm.

To get more definitive results, we observed samples with High Resolution Transmission Electron Microscopy (HRTEM). The following two images are polymerized microtubules observed in the system containing secreted mCherry-α-tubulin and β-tubulin. Several microtubules can be seen on these images.

Figure 10 Electron microscopy images of polymerized microtubules.
A Linear microtubule observed with HRTEM. The red arrows indicate the microtubules;
B Enlarged view of image A. The red arrows indicate the microtubules.

From the results we got with absorbance curves and electron microscopy images, the function of the secrete module can be verified.

Function verification of upgraded display system

As the function of the secrete module was verified, we experimented on the polymerization of microtubules at the surface of the yeast cells. Specifically, we did the polymerization reaction with tubulins extracted from the brain tissue of Sus scrofa domesticus and yeasts displaying β-tubulin. We did microscopic exam with HRTEM. Polymerized microtubules were observed on the cell wall of the yeasts, of which the quantity and lengths were consistent with our model prediction. The followings are the results.

Figure 11 Microscopic images of polymerized microtubules on yeast cell wall.
A Polymerization with tubulins extracted from the brain tissue of Sus scrofa domesticus and yeasts displaying β-tubulin. Considerable numbers of microtubule were displayed on the yeast cell wall;
B Control: Yeasts displaying β-tubulin without free tubulins. There were no observable microtubules on the yeast cell wall.

Figure 12 Enlarged view of microtubules on the yeast surface in Fig. 11A
The red arrows indicate the microtubules.

Figure 13 More enlarged view of microtubules on the yeast surface in Fig. 11A
The red arrows indicate the microtubules.

Figure 14 Microscopic image of a free microtubule, which is obviously longer than those polymerized on yeast surface.

Flagellar Filament

Display module

Plasmid construction

We have successfully constructed 11 parts that have been described in detail in the previous design page. (Flagellar filament module)
In display module, we constructed and validated the following 6 parts. They are pYD1-FliC (BBa_K2220002), pYD1-XynA(BBa_K2220004), pYD1-PETase(BBa_K2220005), pYD1-BG(BBa_K2220007), pYD1-EG(BBa_K2220006), and pYD1-CBH(BBa_K2220008), which means to fuse the target gene sequences with AGA2 gene respectively. We also constructed pYD1-FilC(eGFP) (BBa_K2220003) as our positive control. The length and sequence of each parts have been validated by sequencing and electrophoresis. The length validation are presented on the part registry page.

Protein expression analysis-Fluorescence Microscopy

Recombinant S. cerevisiae EBY100 strain harbouring pYD1-FliC(eGFP) plasmid was precultivated to mid-log growth phase and then induced for 24 hours at 20℃ in SG-CAA medium. The expression of recombinant protein FliC(eGFP) can be obviously observed from the fluorescence microscope field.

Figure 15 Induced for 24h in SG-CAA medium;
A,B Recipient strain with empty plasmid;
C Bright-field micrograph of S. cerevisiae EBY100 cells harbouring pYD1–FliC(eGFP);
D Fluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYD1–FliC(eGFP).

Protein function analysis- Enzyme activity assay

We then validated the function of our pYD1-FliC(XynA) by testing the enzyme activity of recombinant S. cerevisiae EBY100 harbouring pYD1-FliC(XynA). We cultured and induced control group(EBY100-pYD1) and experimental group(EBY100-FliC(XynA)) at the same initial concentration in SG-CAA medium (plus 5% xylan). Supernatant of the same volume were collected every two hours from 10h to 18h to examine the concentration of reducing sugars by DNS method. The results are shown below.

Figure 16 Xylnase enzyme activity assay curve.

The value of OD₅₄₀ is of positive correlation with the concentration of reducing sugar assayed by DNS method. So here we used the value of OD₅₄₀ to estimate the concentration of reducing sugar. Before 14h, engineered yeasts were consuming the galactose in medium, so the OD₅₄₀ values of two groups were similarly decreasing. After 14h, the OD₅₄₀ of the control group was still decreasing, while the values of the experimental group had changed to increase, and decreased again after 16h, which means that FliC(Xylanase) proteins had been displayed in a certain quantity at the beginning of 14h and degraded the xylan of culture medium to xylose, so the concentration of reducing sugar increased. After a period of time, yeast absorption rate of xylose and galactose was greater than that of xylan decomposed by enzyme, so the concentration of reducing sugar decreased again. Thus, from here we conclude that our FilC(XynA) were successfully displayed on the yeast surface.

Secretory module

Plasmid construction

In secretory module,we successfully constructed the following parts: pYCα-FliC(XynA) (BBa_K2220011), pYCα-FliC(BG) (BBa_K2220014), pYCα-FliC(EG) (BBa_K2220013), pYCα-FliC(CBH) (BBa_K2220015), and pYCα-FliC(eGFP) (BBa_K2220003) as positive control. The lengths and sequences of each part have been validated by sequencing and electrophoresis. The length validations are presented on the part registry page. The function of these parts are described on previous design page.

Protein expression analysis- Fluorescence microscopy

Recombinant S. cerevisiae INVSc1 strain harbouring pYCα-FliC(eGFP) plasmid was precultivated to mid-log growth phase respectively and then induced for 10 h at 30℃ in SG-Ura medium. To detect the expression of recombinant protein, we used fluoresence microscope Axio Imager A1 to observe it. As image below shows, the recombinant protein FliC(eGFP) had been expressed and worked as expected.

Figure 17 Induced for 10h in SG-CAA medium;
A,B Recipient strain with empty plasmid;
C Bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–FliC(eGFP);
D Fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–FliC(eGFP).

Protein expression analysis- Western blot

Recombinant S. cerevisiae INVSc1 strain harbouring pYCα-FliC(PETase) or pYCα-FliC(XynA) plasmid was respectively precultivated to mid-log growth phase, and then induced in SG-Ura medium for 24 hours at 30°C. After inducing, the supernatant from whole-cell lysates of the engineered yeasts mentioned above was analysed by Western blot carried out with an anti-His antibody.

Figure 18 The results of the Western blot analysis carried out with an anti-His antibody

Moreover, we also verified the secretion of FliC(XynA) from engineered INVSc1-FliC(XynA) by Western blot analysis and tested it enzyme activity.

Figure 19 Western blot analysis of the supernatant from S. cerevisiae INVSc1 harbouring pYCα-FliC(XynA) culture, carried out with anti-His antibody.

Protein function analysis- Enzyme activity assay

Enzyme activity assay was carried out on purified proteins. After the reaction proceeded at 50°C for 5 mins, the OD value at 540nm was 0.667, which suggested that the concentration of xylose was 0.512mg/mL. The standard curve used for the calculation is shown as follow.

Figure 20 Xylanase activity assay standard curve
xylose standard curve: C = OD×0.7652 + 0.002068, R² = 0.9942

Protein Function analysis- Electron microscopy

We polymerized purified flagellin (Xylanase) secreted by yeasts harbouring the plasmid pYCα-FliC(XynA). The microscopic images on HRTEM, proving that the secreted protein can function as designed, are shown below.

Figure 21 Microscopic images of flagellar filaments polymerized from secreted flagelin with different magnification.
The red arrows indicate the flagellar filaments.