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| + | <h1>Experiments</h1> |
| + | </div> |
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| + | <!--内容--> |
| + | <div class="content-small"> |
| + | <section class="article p-t-54 p-b-54"> |
| + | <section> |
| + | <h1 class="title">Experiment 1: Plasmid transformation</h1> |
| | | |
| + | <section> |
| + | <h2 class="title2">1. Escherichia coli plasmid transformation</h2> |
| + | <p>100μl of <i>Escherichia coli</i> DH5α competent cell which is kept in -80 ℃ was used. After the <i>Escherichia |
| + | coli</i> melts on the ice, plasmid PHT43 was added and incubated on ice for 30 minutes.</p> |
| + | <p>And then, the <i>Escherichia coli</i> with the plasmid was heated in 42 ℃, for 90 seconds, so that |
| + | the coli is able to absorb the plasmid.</p> |
| + | <p>And then, the cells were cooled on ice for another 3 to 4 minutes.</p> |
| + | <p>LB culture medium without antibiotic was added into the <i>Escherichia coli</i> DH5α cells, and then, |
| + | incubated under 37 ℃ for 30 min on the shaker so that DH5α <i>Escherichia coli</i> will be able to |
| + | express protein against ampicillin.</p> |
| + | <p>We then plated the DH5α <i>Escherichia coli</i> to the LA culture medium with antibiotic AMP to check |
| + | if it is able to resist AMP.</p> |
| + | <p>If it grows, it proves that the plasmid was successfully transformed.</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h2 class="title2">2. Plasmid electric transfer to Bacillus subtilis</h2> |
| + | <p>1. Preparation of <i>Bacillus subtilis</i> competent cells</p> |
| + | <p>10 ml LB with sorbitol + 10 µ l B. <i>subtilis</i></p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h2 class="title2">3. Electrotransformation</h2> |
| + | <p>Cooled the <i>Bacillus subtilis</i> on ice for 10 min</p> |
| + | <p>The bacteria were then collected by centrifugation at 5000×g for 5 min at 4°C.</p> |
| + | <p>And then, the suspension was resuspended in 50 ml of precooled electrospun medium, centrifuged at |
| + | 5000 × g for 5 min at 4°C to remove the supernatant.</p> |
| + | <p>Repeat the above process for three times.</p> |
| + | <p>The washed <i>Bacillus</i> were resuspended in 2 ml of the electrospun medium, and each 100 μl was |
| + | transferred into a new EP tube.</p> |
| + | <p>8 μl of plasmid was added to 100 μl of competent cells, incubated on ice for 2 min, and then was |
| + | added into a precooled electric cup.</p> |
| + | <p><i>Subtilis</i> cell walls were electric shocked in a 1 mm cuvette (1 mm gap in the electrode)</p> |
| + | <p>Electric shock preparation, wipe clean the cuvettes without water</p> |
| + | <section style="padding-left: 32px"> |
| + | <p>gap, 1 mm</p> |
| + | <p>200 ohm</p> |
| + | <p>25 μf</p> |
| + | </section> |
| + | <p>Electric shock once</p> |
| + | <p>Experimental group ①: 3.2 MS (eight microliters of plasmid added)</p> |
| + | <p>Experimental group ②: 3.6 MS (8 μl plasmid added)</p> |
| + | <p>Control ①: 3.9 MS (plus 8 μl of water)</p> |
| + | <p>After the electric shock, the cuvettes was removed and the bacteria were immediately added to 1 ml of |
| + | RM (LB + 0.5mol sorbitol + 0.38mol of mannitol)</p> |
| + | <p>After resuscitation at 37°C for 3 h, the bacteria was incubated overnight.</p> |
| + | <p style="color: #0000FF">The <i style="color: #0000FF">E. coli</i> BL21 and the DH5α were bought from |
| + | Beijing Zoman Biotechnology Company</p> |
| + | </section> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experiment 2: PCR</h1> |
| + | <img src="https://static.igem.org/mediawiki/2021/6/6c/T--Shanghai_high_school--img_experiments_1.jpg" alt="" style="width: 90%"> |
| + | <section class="m-t-18 m-b-18" style="padding-left: 48px"> |
| + | <p>The first round PCR</p> |
| + | <p>20 μl of the PCR system</p> |
| + | <p>10 μl of enzyme (purple) Inc.: Vazyme</p> |
| + | <p>Two primers (white pet28a-vp7-ltb (both ends of complete gene sequence)) upstream and downstream, 1μl |
| + | each.</p> |
| + | </section> |
| + | <img src="https://static.igem.org/mediawiki/2021/7/73/T--Shanghai_high_school--img_experiments_2.jpg" alt="" style="width: 90%"> |
| + | <img src="https://static.igem.org/mediawiki/2021/2/23/T--Shanghai_high_school--img_experiments_3.jpg" alt="" style="width: 90%"> |
| + | <p class="m-t-18">Template (blue pET28a-<i>vp7-Ltb</i> intact plasmid) 4 μg dry powder (approx. 1 μl)</p> |
| + | <p>The remaining volume was filled up with water (7μl)</p> |
| + | |
| + | <section> |
| + | <h2 class="title2">1. PCR procedure</h2> |
| + | <section> |
| + | <p>① The DNA was denatured by 95 degrees for 3 min to break the duplex</p> |
| + | <p>② 66 degrees of "annealing" let DNA and "primer" complementary pairing for 1min and cycle 5 |
| + | times</p> |
| + | <p>Cycles of 63 degrees for 1min and cycle 5 times</p> |
| + | <p>15 cycles of 60 degrees</p> |
| + | <p>③ Elongation at 72 degrees, replicating DNA</p> |
| + | <p>Put the target genes and the cut thread genes together and let them combine</p> |
| + | <p>④ Degree 4 preserved, program end</p> |
| + | </section> |
| + | |
| + | <section class="p-t-72"> |
| + | <p>Second round PCR system 50 μl</p> |
| + | <p>The enzyme was given at 25 μl</p> |
| + | <p>2 μl of each primer (4 microliters total)</p> |
| + | <p>Template at 1 μl (the product of the first round of PCR)</p> |
| + | <p>The water added for total system of 20 μl</p> |
| + | <p>Vortex centrifugation</p> |
| + | <img class="m-t-18 m-b-18" src="https://static.igem.org/mediawiki/2021/5/5d/T--Shanghai_high_school--img_experiments_4.jpg" alt="" |
| + | style="width: 60%"> |
| + | <p>Put it into the PCR</p> |
| + | </section> |
| + | </section> |
| + | |
| + | <section> |
| + | <h2 class="title2">2. PCR setup:</h2> |
| + | <section style="padding-left: 32px"> |
| + | <p>95 ° 3:00</p> |
| + | <p>{95 ° 30s</p> |
| + | <p>68 ° 30s</p> |
| + | <p>72 ° 1:00} repeat 5 times</p> |
| + | <p>{95 ° 30s</p> |
| + | <p>66 ° 30s</p> |
| + | <p>72 ° 1:00} repeat 5 times</p> |
| + | <p>{95 ° 30s</p> |
| + | <p>65 ° 30s</p> |
| + | <p>72 ° 1:00}repeat 15 times</p> |
| + | <p>72 Degrees 5:00</p> |
| + | <p>4 ° (preserved)</p> |
| + | </section> |
| + | <br> |
| + | <p style="color: #0000FF">The enzyme was bought from Vazyme Biotech company</p> |
| + | <p style="color: #0000FF">The primers were synthesized by Biosune company</p> |
| + | </section> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experiment 3: Enzymatic digestion</h1> |
| + | <p>Plasmid pET28a used with the BamHI enzyme (a kind of restriction enzyme)</p> |
| + | <p>Two tubes of solution with a total of 40 μl : 30 μl plasmid + 6 μl BamH1+ 4 μl (10%) of Cutsmart (a |
| + | solution to provide a good internal cutting environment).</p> |
| + | <p>Plasmid PHT43 was with the XbaI enzyme (a kind of restriction enzyme),</p> |
| + | <p>Two tubes of solution with a total of 40 μl : 30 μl plasmid + 6 μlXbaI+ 4 μl (10%) of Cutsmart.</p> |
| + | <p>Place the four tubes at 37 degrees for 1h</p> |
| + | <p>And then prepare another 10 μl of uncut control group solution (9 μl of empty loading plasmid puls 1 μl |
| + | of buffer without restriction enzyme )</p> |
| + | <br> |
| + | <p style="color: #0000FF">Dye buffer [cutsmart], BamHI, and XbaI were all bought from company NEB (New |
| + | England Biolabs)</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experiment 4: Plasmid extraction</h1> |
| + | <p> (Note: The centrifuge speed has always been 12,000 rpm.</p> |
| + | <p>3ml <i>Bacillus subtilis</i> was pured by centrifugation for 1 min</p> |
| + | <section> |
| + | <p>1. plus 250 μl Solution I (degrading RNA for 2 min)</p> |
| + | <p>2. plus 250 μl Solution II. Lysis the cell wall for 4 min</p> |
| + | <p>3. 350 μl Solution III was added and mixed .The liquid (plasmid solution) was centrifuged after 10 |
| + | min.</p> |
| + | <p>4. centrifugation and the remaining cell body were discarded</p> |
| + | <p>5. loaded the silicon basement membrane again centrifuged and absorbed DNA, then pour down the waste |
| + | liquid. The unrelated compounds on the basement membrane were washed away with ethanol (pour 500μl |
| + | of ethanol on the membrane and then put into a centrifuge for 1 min) leaving only plasmid DNA</p> |
| + | <p>6. Centrifuge for 2 min, and volatilize alcohol at room temperature for 10 min</p> |
| + | <p>7. Using an eluent of 60 degrees to shed the plasmid better from the basement membrane. (40 μl |
| + | eluent)</p> |
| + | <p>8. Put into a centrifuge for 30s</p> |
| + | </section> |
| + | |
| + | <p>Extracted plasmid concentration was measured</p> |
| + | |
| + | <section style="padding-left: 32px"> |
| + | <p>① 117.0 ng / μl L 1.82 2.09</p> |
| + | <p>② 130.9 ng / μl L 1.80 2.01</p> |
| + | <p>③ 169.1 ng / μl 1.77 1.92</p> |
| + | <p>④ 194.5 ng / μl 1.83 2.17</p> |
| + | <p>⑤ 170.1 ng / μl L 1.86 2.33</p> |
| + | <p>⑥ 170.9 ng / μl L 1.77 1.90</p> |
| + | </section> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experiment 5: DNA agarose gelelectrophoresis</h1> |
| + | <p>Agarose Gels:</p> |
| + | <p>0.6g agarose + water = 60ml</p> |
| + | <p>Heat medium heat to boiling, take out and wait until it becomes clear and cool to 60 ° C.</p> |
| + | <p>Add the nucleic acid dye 6 μl</p> |
| + | <p>Voltage: 110 V</p> |
| + | <p>Time: 30 min</p> |
| + | <p>Current: 40 mA</p> |
| + | <br> |
| + | <p style="color: #0000FF">Agarose Gels (Gene Company)</p> |
| + | <p style="color: #0000FF">Nucleic acid dye (擎科生物公司TSINGKE)</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experiment 6: Expression of proteins</h1> |
| + | <p>IPTG was added to 1 ml BL21 with a OD600 concentration of 1 mol/ml.</p> |
| + | <p>A total of 25 samples were obtained.</p> |
| + | <p>Proteins were extracted from 12 IPTG induced BL21 samples as well as three BL21 samples from control |
| + | groups without IPTG addition.</p> |
| + | <br> |
| + | <p>IPTG dosage: 0.5 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (control group) 1-1</p> |
| + | <p>IPTG dosage: 1.0 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (control group) 2-1</p> |
| + | <p>IPTG dosage: 2.0 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (control group) 3-1</p> |
| + | <p>IPTG dosage: 0.5 nmol growth time: 2 h 4 h 5 h 6 h and 0 h (no IPTG control group for <i>E. coli</i> |
| + | vp7-LTB) 1-1</p> |
| + | <p>IPTG dosage: 2.0 nmol growth time: 2h 4h 5h 6h and 0h (no IPTG control group for <i>E. coli</i> vp7-LTB) |
| + | 3-1</p> |
| + | <br> |
| + | <p style="color: #0000FF">IPTG was bought from BBI LIFE SCIENCES CORPORATION</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experiment 7: Purify protein</h1> |
| + | <p>Twenty samples with 150 μl IPTG induced bacteria solution were processed for protein purification.</p> |
| + | <p>There is a lysozyme that can help us get our proteins.</p> |
| + | <p>1. 5,000*g (7,500 rpm) centrifugation for 10 min to collect bacterial cell precipitation.</p> |
| + | <p>2. Add 0.5 ml of Extraction Reagent (0.1% DTT and 1% PMSF in ultrapure water) to every 100 mg of wet |
| + | weight bacteria. Put them on the shaking table at 280 rpm room temperature for 20 min.</p> |
| + | <p>3. Centrifuge in a centrifuge of 15,000 g (13,000 rpm) at 4 ℃ for 5 min, collect the protein supernatant |
| + | as the soluble part of bacteria, and keep the insoluble cell fragments and the precipitation of possible |
| + | inclusion bodies for analysis or recovery. Repeat this step for 3 times.</p> |
| + | <p style="color: #0000FF">The PMSF, DTT, DNase I, Lysozome lysis buffer are all bought from Sangon Biotech</p> |
| + | </section> |
| + | </section> |
| + | </div> |
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