Part:BBa_K4349007

PROMOTER + RBS + NSP4 + ENDOCHI + HISTAG + TERMINATOR

UFMG_UFV_Brazil team designed this composite part to express and secrete PCCHI44 endochitinase from Pochonia chlamydosporia in E. coli. The construct consists of a strong constitutive promoter, RBS, signal peptide NSP4, PCCHI44 endochitinase, Histidine tag(6x) and terminator for E.coli. The composite part is shown below:

Usage and Biology

UFMG_UFV_Brazil synthesized this construction via IDT Grant, receiving it as linear double-strand DNA. We added 3' adenine overhangs with Taq polymerase and proceeded to TA cloning, inserting the construct in pCR™2.1-TOPO™ cloning vector from ThermoFisher (the vector comes with thymine overhangs). Then, we transformed DH5alpha E.coli with the resultant plasmid.

Transformed DH5alpha with pCR™2.1-TOPO™ cloning vector + BBa_K4349007 construct. Colonies are shown inside the drawn circles.

Transformed DH5alpha with pCR™2.1-TOPO™ cloning vector + BBa_K4349007 construct. Colonies are shown inside the drawn circles.

The bacteria grew and we performed Miniprep to obtain more quantity of the plasmid. After that, we performed two digestions (in the Miniprep and in the pSB1C3): first with EcoRI and then with PstI. We did the digestions and the ligation to insert our construct in the expression vector pSB1C3 from iGEM.

EcoRI digestion

The first digestion with EcoRI was confirmed in this 1% agarose gel electrophoresis. We expected to obtain a digested endochitinase construct with 1241bp. The gel has some stains because of RNA contamination.

Endo represents the BBa_K4349007 construct and (NA1) represents the bacterium colony where the sample was originated.

PstI digestion
We extracted the digested endochitinase construct and the digested pSB1C3 from the EcoRI gel. We digested the resultant samples with PstI. Then, we precipitated the post-digestion DNA samples.

Ligation
After that, we ligated the backbone + the insert with the T4 DNA Ligase. We left the reaction in the Thermocycler 16°C overnight.

BL21 Transformation
After inserting BBa_K4349007 in the expression vector pSB1C3, we transformed BL21 E.coli. After they grew, we did another Miniprep.

Transformed BL21 with pSB1C3 expression vector + BBa_K4349007 construct, 100ul. Colonies are shown inside the drawn circles.

Transformed BL21 with pSB1C3 expression vector + BBa_K4349007 construct, 200ul. Colonies are shown inside the drawn circles.

NotI digestion

We checked if the insertion in pSB1C3 was successful by digesting the resultant Miniprep with NotI. The restriction sites for NotI are present in the Biobrick prefix and suffix. We observed the expected bands in the 1% gel electrophoresis: 1181 bp - Endo A1 and Endo B1.

Endo represents the BBa_K4349007 construct and the codes represent the bacterium colony where the samples were originated.

We also proved that the insertion was successful by doing PCR with the primers VF2 and VR.

Endo represents the BBa_K4349007

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 89
  Illegal NgoMIV site found at 720
  
• 1000
  COMPATIBLE WITH RFC[1000]