Part:BBa_K4127021

Cas12a Protein Plasmid

Under the cooperation discussion, our partner NUDT_CHINA helped us design the expression plasmid of Cas12a protein

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1147
  Illegal BglII site found at 1768
  Illegal BglII site found at 2035
  Illegal BglII site found at 2605
  Illegal BglII site found at 2938
  Illegal BglII site found at 4129
  Illegal BamHI site found at 3942
  Illegal XhoI site found at 158
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1175
  Illegal NgoMIV site found at 1877
  Illegal NgoMIV site found at 2876
  Illegal NgoMIV site found at 4161
  Illegal NgoMIV site found at 5749
  Illegal NgoMIV site found at 5909
  Illegal NgoMIV site found at 8956
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1901
  Illegal BsaI site found at 2261
  Illegal BsaI site found at 2441
  Illegal BsaI site found at 3503
  Illegal SapI site found at 995
  Illegal SapI site found at 1615
  Illegal SapI site found at 1635
  Illegal SapI site found at 2486
  Illegal SapI site found at 2546
  Illegal SapI site found at 6829
  

Source:

The Echinococcus characteristic sequences, 70 to 90 bp in length, are from the Echinococcus genome.

1.Expression vector and resistance

pET-28a（Kanamycin）

2.plasmid profile

Figure 1:Cas12a Protein Plasmid 9097bp

3.Complete sequence of the target protein

>LbCpf1
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYI
SLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRE
NMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYN
AIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSI
KKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFS
LEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFF
GEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRY
GSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKG
DMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLY
MFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPK
KTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVE
QYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGF
KNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDP
STGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVF
DWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSD
GIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH

4．Expression and purification strategies

⚫ Cas12a expression plasmids were transformed into E. coli BL21 (DE3) (Table S5).
⚫ For protein expression, a single clone was first cultured overnight in 5-mL liquid LB tubes and then 1% (v/v) inoculated into 3 L of fresh liquid LB. Cells were grown with shaking at 220 rpm and 37 °C until the OD600 reached 0.8, and IPTG was then added to a final concentration of 0.2 mM followed by further culture of the cells at 16 °C for about 16 h before the cell harvesting.
⚫ Cells were resuspended in 50 mL of lysis buffer [50 mM Tris-HCl (pH 8.0), 1.5 M NaCl, 1 mM DTT and 5% glycerol] with 1 mM PMSF as the protease inhibitor and lysed by high pressure.
⚫ The obtained lysate was then centrifuged twice at 15,000 rpm for 30 min. After centrifuging, the supernatant was mixed with 5 mL of Ni-NTA beads (GE Healthcare) and softly shaken for 1 h at 4 °C before being loaded onto a 30-mL column.
⚫ The packing was then washed with wash buffer (lysis buffer supplemented with 30 mM imidazole) and eluted with elution buffer (lysis buffer supplemented with 600 mM imidazole). The elution was dialysed with dialysis buffer 1 [50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM DTT and 5% glycerol].
⚫ Before the protein solution was loaded onto an anion exchange column (HiTrapTM Q HP, GE Healthcare), it was diluted until the final NaCl concentration reached below 80 mM. After that, the column was washed and then eluted with a gradient concentration of NaCl (AKTA Pure, GE Healthcare).
⚫ Fractions containing Cas12a proteins were verified by SDS-PAGE and then pooled for dialysis with dialysis buffer 2 [20 mM Tris-HCl (pH 8.0), 600 mM NaCl, 2 mM DTT, 0.2 mM EDTA] overnight.
⚫ Finally, the protein was collected and diluted to a final concentration of 6 μM and mixed with an equal volume of 100% cold glycerol prior to being frozen at −80 °C.