Difference between revisions of "Team:SCUT-ChinaA/Experiments"

Line 58: Line 58:
 
</li><li>2.    Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
 
</li><li>2.    Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
 
</li><li>3.    Add 1 ml EZ 2 solution to resuspend the pellet.
 
</li><li>3.    Add 1 ml EZ 2 solution to resuspend the pellet.
 +
</li>
 +
</ul>
 +
<p>At this point, the competent cells can be used for transformations directly or stored frozen at or below -70℃ for future used. It is important to freeze the cells slowly. To accomplish this, either wrap the aliquoted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer. Do not use liquid nitrogen to snap-freeze the cells.
 +
</p><p>With yeast competent cells, we can then carry out yeast transformation.
 +
</p>
 +
<ul>
 +
<li>1.    Mix 50 μl of competent cells with 0.2-1 μg DNA (in less than 5 μl volume); add 500 μl EZ 3 solution and mix thoroughly.
 +
</li><li>2.    Incubate at 30℃ for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
 +
</li><li>3.    Spread 50-150 μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.
 +
</li><li>4.    Incubate the plate at 30℃ for 2-4 days to allow for growth of transformations.
 
</li>
 
</li>
 
</ul>
 
</ul>
  
<div class="column half_size" >
 
<div class="highlight decoration_A_full">
 
<img class="full_size_image" src="https://static.igem.org/mediawiki/2018/c/c8/T--SCUT-ChinaA--p3.png">
 
</div>
 
<figcaption><h6>Figure 3: the pathway design of limonene synthesis. The yellow arrow represents the rate-limiting enzymes (tHMG1 and ERG12). The orange arrow represents the enzymes (NDPS1 and LS) heterologously expressed in <em>Y. lipolytica</em>.</h6> </figcaption>
 
</div>
 
  
<h3>2. Overexpression</h3>
+
<h7>Yeast total RNA Extraction</h7>
<p>Enhancing the gene copy number of the enzyme on the synthetic pathway, might lead to improvements of the limonene production. For further optimizing the flow of metabolic flux, we decided to overexpress the rate limiting enzymes involved in the limonene synthesis pathway, including tHMG1, ERG8, ERG12 and ERG19. The tHMG1 gene was cloned from the chromosome of the <em>Y. lipolytica</em>, while the ERG8, ERG12 and ERG19 with their terminators were cloned from the genome. Moreover, to increase the application scope of overexpression modules, we assembled the expression cassettes of ERG8/12/19 together for the overexpression of other metabolic pathway. Homologous tHMG1 gene from <em>Y. lipolytica</em> was overexpressed under the control of GPD2 promoter in the limonene-producing strain Po1f/lim. ERG8/12/19 was fused with EXP1, TEF1 and GPD2 promotor, respectively. Then the tHMG1 expression cassettes fused with ku80upstream/downstream was transformed into Po1f/lim with the plasmid pCAS1yl-ku80, simultaneously achieve the purpose of knocking out ku80 gene and introducing tHMG1 gene. The ERG8/12/19 module was integrated into the pox5 site of the genome synchronously, obtaining engineered strain named Po1f/lim-tE.
+
<p>Most of RNA in the cell is bound to protein and exists as nuclear protein. Therefore, RNA is separated and removed from protein when extracting RNA. The cells are placed in a buffer containing sodium dodecylsulfonate, and then water-saturated phenol and chloroform are added to it. Through vigorous shaking and centrifuging, the liquid is divided into the upper aqueous phase and the lower phenol phase. The nucleic acid is dissolved in the aqueous phase, the protein denatured by the phenol is either dissolved in the phenol phase, or forming a gel layer at the interface of the two phases. The upper aqueous phase is collected and the RNA is precipitated from the aqueous solution with ethanol.
 +
<p></p>All necessary materials for the experiment should be prepared as described below:
 
</p>
 
</p>
 +
<ul>
 +
<li>1.    0.2% DEPC-treated water: Wash and dry a wide-mouth bottle, wash it with hydrogen peroxide for 3 times, and then wash it with absolute ethanol for 3 times. After drying, add 1 L ultrapure water, then add 2 ml DEPC in a fume hood, shake overnight at 37 °C, sterilize at 121 °C for 30 min, and then store at room temperature.
 +
</li><li>2.    AE buffer (pH 5.2, 50 mM NaAc, 10 mM EDTA): Weigh 0.41 g anhydrous NaAc, 0.37 g EDTA Na2, dissolve in 80 mL pure water, adjust the pH to 5.2 with glacial acetic acid, and then constant volume to 100 mL. Add 200 μl DEPC, shake overnight at 37 °C, sterilize at 121 °C for 45 min, and then store at room temperature.
 +
</li><li>3.    10% (w/v) SDS: Weigh 10 g SDS, dissolve with 80 mL DEPC-treated water, and constant volume to 100 mL with DEPC-treated water, don’t need sterilization. Store at room temperature.
 +
</li><li>4.    Water-saturated phenol:chloroform (1:1): While taking water-saturated phenol, the tips should be inserted into the underlying organic phase. Then mix with chloroform and store. If the upper layer still has an aqueous phase, use pipettes to remove it.
 +
</li><li>5.    3M NaAc (pH 5.2): Weigh 24.6 g anhydrous NaAc, dissolve in 80 mL pure water, adjust the pH to 5.2 with glacial acetic acid, constant volume to 100 mL, add 200 μl DEPC, shake overnight at 37 °C, 121 °C Sterilize for 30 min and store at room temperature.
 +
</li><li>6.    Absolute ethyl alcohol
 +
</li><li>7.    75% (v/v) ethanol: Mix 1 volume of DEPC water with 3 volumes of absolute ethanol.
 +
</li><li>8.    0.2% DEPC treated TAE buffer: Measure out 20 mL TAX buffer, add DEPC treated water to 1 L, shake at 37 °C overnight (ensure to fix the bottle, prevent overflow)
 +
</li>
 +
</ul>
 +
 +
<p>The specific experimental steps are as mentioned follows:</p>
 +
<ul>
 +
<li>1.    Cell collection: Place 10 mL YPD in a 50 mL centrifuge tube and inoculate Yarrowia lipolytica for 16-18 h to an OD of 5-7. Collect 10 OD cells in a 1.5 mL centrifuge tube, centrifuged at 10000 rpm for 2 min, discard the supernatant. Centrifuge briefly and remove the remaining medium with a pipette. Quickly freeze the cells in a liquid nitrogen tank and store at -80 °C refrigerator (can be stored for 2-3 days).
 +
</li><li>2.    Take and place the cells sample on ice, resuspend the cells in 50 μl AE buffer and mix by pipetting.
 +
</li><li>3.    Take a 1.5 mL centrifuge tube and draw 600 μL phenol: chloroform (1:1), 350 ul AE buffer, and 50 ul SDS into it, quickly mix upside down and preheat in 65 °C metal bath for 5 minutes.
 +
</li><li>4.    After incubation, incubate the cells at 30 °C, 250 rpm for 3 days, measure the OD and collect 10 OD bacteria. Centrifuge the cells at 4 °C,12000 g for 2min, remove the supernatant, add the mixture in step 3 to it, quickly reverse three times to mix well, immediately put into a 65 °C metal bath, and shake it at 1000 rpm for 14 min, and ice bath for 5 min.
 +
</li><li>5.    Centrifuge at 4 °C, 12000 g for 7min, softly take 300 ul supernatant into a 1.5 mL centrifuge tube containing 750 ul absolute ethanol and 30 ul 3M NaAc (pH 5.2), mix upside down, incubate at -20℃ for more than 30 min.
 +
</li><li>6.    Centrifuge at 4 °C, 12000 g for 5min, remove the supernatant with a 200ul pipette (be careful not to aspirate the RNA pellet), add 1 mL 75% ethanol to wash the pellet (Flick the tube bottom by finger, upside down ten times).
 +
</li><li>7.    Centrifuge at 4 °C, 12000 g for 3min, adjust the pipette to 500ul to remove the supernatant (Be careful not to aspirate the RNA pellet). Centrifuge for about 30 s and adjust the pipette to 50 ul to remove the supernatant (Be careful not to aspirate the RNA pellet). Store at room temperature for 3 min to dry, add 37.5 ul DEPC treated water to dissolve the precipitate (The precipitate on the side wall of the centrifuge tube should also be dissolved).
 +
</li><li>8.    Use Nanodrop ultra-micro UV spectrophotometer to determine RNA concentration and parameters.
 +
</li><li>9.    Take 1 ug for electrophoresis. 25-40min, 60-80v. The running comb, rubber tray, rubber plate and electrophoresis tank all should be soaked in disinfectant for 20 minutes and rinsed once with absolute ethanol. TAE buffer should be treated with DEPC-treated water.
 +
 +
</li>
 +
</ul>
 +
 +
<p>Here are some points that need special attention.</p>
 +
<ul>
 +
<li>
 +
1.    The water for preparing solution is DEPC-treated water.
 +
</li><li>2.    Wear latex gloves and replace them frequently during the entire operation to avoid RNase contamination. Wear a mask and talk as little as possible.
 +
</li><li>3.    Remove the lower phenol phase when using water-saturated phenol.
 +
</li><li>4.    Carefully remove the upper aqueous phase and do not absorb the denaturing gel layer between the aqueous phase and the phenolic phase.
 +
</li>
 +
</ul>
  
<h3>3. Scaffold Application</h3>
 
<p>
 
The SpyTag/SpyCatcher system has a wide range of applications in enhancing the expression of metabolic pathways and rerouting pathways. We applied SpyTag/SpyCatcher tagging system to develop a high-performance enzyme self-assembling system (HESS) to pull the metabolic flux to NPP and limonene instead of GPP. Considering the size of the proteins structure, we attached SpyCatcher to NDPS1 and SpyTag to LS. On the other hand, we fused SpyCatcher to the C terminal of NDPS1 and SpyTag to the N terminal of LS for ensuring the catalytic activity of the enzymes. So, based on the co-expression module, which contents the expression cassettes of NDPS1 and LS, we developed the HESS to enhance the flow of metabolic pathway. The SpyCatcher gene was cloned into the module between the NDPS1 and XPR terminator, while the SpyTag gene was cloned into the module between the EXP promoter and LS gene. The HESS module would be integrated into the rDNA site of the chromosome in the Po1f/lim-tE to get the strain Po1f/lim-tESS for fermentation.
 
</p>
 
  
 
</ol>
 
</ol>

Revision as of 05:50, 17 October 2018

Construction

    1.Design

    In our project, for obtaining various engineered strains, we applied module assembly technology like gibson assembly and overlap extension PCR to construct gene expression modules in E. coli. In the process of pathway construction, we successfully constructed pUC19-NDPS1-LS, pUC19-Catcher-NDPS1-Tag-LS, pUC19-tHMG1, pUC19-ERG8-ERG12-ERG19, pCAS1yl-ku70, pCAS1yl-pox5, respectively. The upstream and downstream regions are homologous to the target integration site and prepared for homologous recombination in further gene editing. The LEU2 and URA3 was used for selection in Y. lipolytica and the AmpR was used in E.coli. NotI restriction sites was added between the homology regions. These plasmids will be further linearized in the subsequent steps.

    2.Method

    Gibson assembly

    The method can simultaneously combine up to 15 DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. These DNA fragments are mixed with a cocktail of three enzymes, along with other buffer components. The three required enzyme activities are: exonuclease, DNA polymerase, and DNA ligase. The exonuclease chews back DNA from the 5' end, thus not inhibiting polymerase activity and allowing the reaction to occur in one single process. The resulting single-stranded regions on adjacent DNA fragments can anneal. The DNA polymerase incorporates nucleotides to fill in any gaps. The DNA ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA.

    The detailed protocol is as follows:

    • 1. Add overlap region to adjacent DNA fragments via PCR;
    • 2. Combine the to-be-assembled DNA to a total volume of 5 μl;
    • 3. On ice, add 15μl Master Mix to the DNA, mix well and briefly centrifuge;
    • 4. Incubate at 50℃ for 1 hour;
    • 5. Store reactions at -20℃ or proceed to transformation.

Integration

    1.Design

    After the successfully construction of various expression modules, we linearized them with NotI and then were transformed into competent Po1f cells together, using the kit, Frozen-EZ yeast transformation II. After transformation, cells were cultured on SD medium plates without uracil or leucine. The URA3 marker was integrated into the cells along with the expression cassettes, which would affect the insertion of subsequent gene expression modules. Therefore, YPD media supplemented with 1 mg/mL 5-FOA was used for URA3 marker plasmid removal. Screening for integration was accomplished using colony PCR of single colonies. Furthermore, we would further verify it by extracting the genome of the yeast, using the kit from TIANGEN Biotech (Beijing, China). At the transcriptional level, we verified the transcription of the integrated gene modules by quantitative PCR with a kit from XXXXX.

    2. Method

    Transformation

    First, prepare the yeast competent cells. Grow yeast cells at 30℃ in 10ml YPD broth until mid-log phase (OD600 of 0.8-1.0). The following steps are accomplished at room temperature.

    • 1. Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
    • 2. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
    • 3. Add 1 ml EZ 2 solution to resuspend the pellet.

    At this point, the competent cells can be used for transformations directly or stored frozen at or below -70℃ for future used. It is important to freeze the cells slowly. To accomplish this, either wrap the aliquoted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer. Do not use liquid nitrogen to snap-freeze the cells.

    With yeast competent cells, we can then carry out yeast transformation.

    • 1. Mix 50 μl of competent cells with 0.2-1 μg DNA (in less than 5 μl volume); add 500 μl EZ 3 solution and mix thoroughly.
    • 2. Incubate at 30℃ for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
    • 3. Spread 50-150 μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.
    • 4. Incubate the plate at 30℃ for 2-4 days to allow for growth of transformations.
    Yeast total RNA Extraction

    Most of RNA in the cell is bound to protein and exists as nuclear protein. Therefore, RNA is separated and removed from protein when extracting RNA. The cells are placed in a buffer containing sodium dodecylsulfonate, and then water-saturated phenol and chloroform are added to it. Through vigorous shaking and centrifuging, the liquid is divided into the upper aqueous phase and the lower phenol phase. The nucleic acid is dissolved in the aqueous phase, the protein denatured by the phenol is either dissolved in the phenol phase, or forming a gel layer at the interface of the two phases. The upper aqueous phase is collected and the RNA is precipitated from the aqueous solution with ethanol.

    All necessary materials for the experiment should be prepared as described below:

    • 1. 0.2% DEPC-treated water: Wash and dry a wide-mouth bottle, wash it with hydrogen peroxide for 3 times, and then wash it with absolute ethanol for 3 times. After drying, add 1 L ultrapure water, then add 2 ml DEPC in a fume hood, shake overnight at 37 °C, sterilize at 121 °C for 30 min, and then store at room temperature.
    • 2. AE buffer (pH 5.2, 50 mM NaAc, 10 mM EDTA): Weigh 0.41 g anhydrous NaAc, 0.37 g EDTA Na2, dissolve in 80 mL pure water, adjust the pH to 5.2 with glacial acetic acid, and then constant volume to 100 mL. Add 200 μl DEPC, shake overnight at 37 °C, sterilize at 121 °C for 45 min, and then store at room temperature.
    • 3. 10% (w/v) SDS: Weigh 10 g SDS, dissolve with 80 mL DEPC-treated water, and constant volume to 100 mL with DEPC-treated water, don’t need sterilization. Store at room temperature.
    • 4. Water-saturated phenol:chloroform (1:1): While taking water-saturated phenol, the tips should be inserted into the underlying organic phase. Then mix with chloroform and store. If the upper layer still has an aqueous phase, use pipettes to remove it.
    • 5. 3M NaAc (pH 5.2): Weigh 24.6 g anhydrous NaAc, dissolve in 80 mL pure water, adjust the pH to 5.2 with glacial acetic acid, constant volume to 100 mL, add 200 μl DEPC, shake overnight at 37 °C, 121 °C Sterilize for 30 min and store at room temperature.
    • 6. Absolute ethyl alcohol
    • 7. 75% (v/v) ethanol: Mix 1 volume of DEPC water with 3 volumes of absolute ethanol.
    • 8. 0.2% DEPC treated TAE buffer: Measure out 20 mL TAX buffer, add DEPC treated water to 1 L, shake at 37 °C overnight (ensure to fix the bottle, prevent overflow)

    The specific experimental steps are as mentioned follows:

    • 1. Cell collection: Place 10 mL YPD in a 50 mL centrifuge tube and inoculate Yarrowia lipolytica for 16-18 h to an OD of 5-7. Collect 10 OD cells in a 1.5 mL centrifuge tube, centrifuged at 10000 rpm for 2 min, discard the supernatant. Centrifuge briefly and remove the remaining medium with a pipette. Quickly freeze the cells in a liquid nitrogen tank and store at -80 °C refrigerator (can be stored for 2-3 days).
    • 2. Take and place the cells sample on ice, resuspend the cells in 50 μl AE buffer and mix by pipetting.
    • 3. Take a 1.5 mL centrifuge tube and draw 600 μL phenol: chloroform (1:1), 350 ul AE buffer, and 50 ul SDS into it, quickly mix upside down and preheat in 65 °C metal bath for 5 minutes.
    • 4. After incubation, incubate the cells at 30 °C, 250 rpm for 3 days, measure the OD and collect 10 OD bacteria. Centrifuge the cells at 4 °C,12000 g for 2min, remove the supernatant, add the mixture in step 3 to it, quickly reverse three times to mix well, immediately put into a 65 °C metal bath, and shake it at 1000 rpm for 14 min, and ice bath for 5 min.
    • 5. Centrifuge at 4 °C, 12000 g for 7min, softly take 300 ul supernatant into a 1.5 mL centrifuge tube containing 750 ul absolute ethanol and 30 ul 3M NaAc (pH 5.2), mix upside down, incubate at -20℃ for more than 30 min.
    • 6. Centrifuge at 4 °C, 12000 g for 5min, remove the supernatant with a 200ul pipette (be careful not to aspirate the RNA pellet), add 1 mL 75% ethanol to wash the pellet (Flick the tube bottom by finger, upside down ten times).
    • 7. Centrifuge at 4 °C, 12000 g for 3min, adjust the pipette to 500ul to remove the supernatant (Be careful not to aspirate the RNA pellet). Centrifuge for about 30 s and adjust the pipette to 50 ul to remove the supernatant (Be careful not to aspirate the RNA pellet). Store at room temperature for 3 min to dry, add 37.5 ul DEPC treated water to dissolve the precipitate (The precipitate on the side wall of the centrifuge tube should also be dissolved).
    • 8. Use Nanodrop ultra-micro UV spectrophotometer to determine RNA concentration and parameters.
    • 9. Take 1 ug for electrophoresis. 25-40min, 60-80v. The running comb, rubber tray, rubber plate and electrophoresis tank all should be soaked in disinfectant for 20 minutes and rinsed once with absolute ethanol. TAE buffer should be treated with DEPC-treated water.

    Here are some points that need special attention.

    • 1. The water for preparing solution is DEPC-treated water.
    • 2. Wear latex gloves and replace them frequently during the entire operation to avoid RNase contamination. Wear a mask and talk as little as possible.
    • 3. Remove the lower phenol phase when using water-saturated phenol.
    • 4. Carefully remove the upper aqueous phase and do not absorb the denaturing gel layer between the aqueous phase and the phenolic phase.

Reference