Part:BBa_K4127002

E gene

We designed expression plasmids of related genes based on the known E gene sequences in the database. It can be expanded in E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 477
  Illegal EcoRI site found at 657
  Illegal XbaI site found at 484
  Illegal PstI site found at 35
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 477
  Illegal EcoRI site found at 657
  Illegal PstI site found at 35
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 477
  Illegal EcoRI site found at 657
  Illegal BamHI site found at 53
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 477
  Illegal EcoRI site found at 657
  Illegal XbaI site found at 484
  Illegal PstI site found at 35
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 477
  Illegal EcoRI site found at 657
  Illegal XbaI site found at 484
  Illegal PstI site found at 35
  
• 1000
  COMPATIBLE WITH RFC[1000]

Source:

The characteristic sequence of E gene, from the COVID-19 genome.

Figure1:E gene 763bp

1.Heat shock transformation of Escherichia coli

⚫ Melt the competent Escherichia coli on the ice.
⚫ Blend the plasmid with the competent Escherichia coli,then take an ice bath for 30 minutes.
⚫ 42°C Heat for 90 s,then take an ice bath for 5 minutes.
⚫ Add 500μl nonresistant LB and put it into a 37°C rocker for 40 minutes.
⚫Centrifuge under 7000 rpm for 1 minute.
⚫ Inhale the upper clearance and leave 40 μl heavy suspension.
⚫ Coating the board with AMP.

2. mini shaking of the plasmid

Select a single coenobium, lift it with the tip,and put it into mixture mixed by 2 ml LB and 2μl AMP,then cultivate the cells by a 37°C 180 rpm rocker for 10 hours.

3. large shaking of the plasmid

Cultivate the mixture mixed by 500 ml LB，500 μl AMP and 1ml shaked plasmid into a 37°C 180 rpm rocker for 16 hours.

4. plasmid mini extraction

⚫ Centrifuge under 8000 rpm for 5 minute,then collect the cells.
⚫ Remove the supernatant and add the 250 μl Buffer P1 / RNase A mixture.
⚫ Add 250 μl Buffer P2,invert to mix and place at ordinary temperature.
⚫ Add 250 μl Buffer P2,invert to mix.
⚫ Centrifuge under 13000 g for 10 minute.
⚫ Place the HiPure DNA Mini ColumnⅡinto a collection tube,then transfer the supernatant to a column and centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and add 500 μl Buffer PW1,then centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and add 600 μl Buffer PW2,then centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and add 300 μl Buffer PW2,then centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and centrifuge under 13000 g for 1 minute.
⚫ Place the column into the collection tube and add 50 μl DEPC water,then centrifuge under 13000 g for 3 minute.
⚫ Collect the filtrate.

5 .plasmid large extraction

⚫ Add 300 ml shaked bacterial liquid into 500 ml tube.
⚫ Centrifuge under 4000 rpm for 15 minute.
⚫ Add 10 ml SolutionⅡ , mix and place at ordinary temperature for 2 min.
⚫ Add 5 ml pre-cooled N3 Buffer,mix and place at ordinary temperature for 2 min.
⚫ Discard the precipitation,then transfer the filtrate to a new 50 ml tube.
⚫ Add 0.1 x volume of ETR Solution.
⚫ Place on the ice 10 min.
⚫ 42℃ water bath for 5 min.
⚫ Centrifuge at 8000 g for 5 min at 25℃.
⚫ Transfer the supernatant to a new 50 ml tube with 0.5 x volume of absolute ethanol,mix and place at ordinary temperature for 2 min.
⚫ Place the DNA Maxi Column into a 50 m l tube,add 3 ml GPS Buffer and place at ordinary temperature for 2 min,then centrifuge at 4000 g for 3 min .
⚫ Discard the filtrate ,add 20 ml of the above-added absolute ethanol solution, and centrifuge at 5,000 g for 3 min.
⚫ Readd the filtrate to DNA Maxi Column and centrifuge at 5,000 g for 3 min.
⚫ Repeat the upper two-step process..
⚫ Add 10 ml HBC Buffer and centrifuge at 5000 g for 3 min.
⚫ Discard the filtrate,add 15 ml DNA Wash Buffer and centrifuge at 5000 g for 3 min.
⚫ Discard the filtrate,add 10 ml DNA Wash Buffer and centrifuge at 5000 g for 3 min.
⚫ Discard the filtrate,then centrifug at 5,000 g for 10 min.
⚫ Remove the DNA Maxi Column and air for 5 min.
⚫ Transfer the DNA Maxi Column to a new 50 ml tube,add 500 ml of sterile water to DNA Maxi Column and place at ordinary temperature for 2 min,then centrifuge at 10,000 g for 10 min,collect the filtrate.
⚫ Repeat the previous step three times.