Designed by: John Anderson   Group: Arkin Lab   (2006-08-04)

BBa_J61004 short

Constitutive expression of invasin under the control of a Tet promoter in a p15A CmR plasmid

Plasmid available by request only to Adam P. Arkin, Christopher A. Voigt, or J. Christopher Anderson.

#Anderson pmid=16330045 Sequence and Features BBa_J61004 SequenceAndFeatures

Contribution by Team:NYU_Abu_Dhabi

Construction and function of the plasmid pAC-TetInv

pAC-TetInv is a medium copy plasmid under the control of a tet promoter.

The first step in the construction of the plasmid pAC-TetInv consists in PCR amplifying the inv gene (GenBank accession no. M17448) from Y. pseudotuberculosisgenomic DNA with oligonucleotides ca783F (5'-CTGAAGGATCCGTTTGACGTATGACAGGTATGC-3') and Rinv (5'-AGGGTCGAATTCTTATATTGACAGCGCACAGA-3'). In order for these two enzymes to be inserted into similar sites of Pac581, they need to be digest with BamHI and EcoRI, The components of the plasmid are mainly a TmBterimnato derived from pBAD/Myc-HisA, atetpromoter, a chloramphenicol resistance gene, and most importantly a p15A origin of replication.

Regarding the lux operon, it was assembled from plasmids pKE705 and pKE555 by first using Xhol and Xbal for digestion then by ligating the e580 bp fragment to a 2980 bp XbaI/BglII fragment of pKE555. PCR with oligonucleotides ca742F (5'-CAGTCGGATCCTTAATTTTTAAAGTATGGGCAATC-3') and ca721R (5'-CACTGGAATTCGTAATGACAGATAATTTTACTC-3') was used to amplify the ligation product. Moreover, BamHI and Ecori enabled the digestion of the full-length of lux operon gene enabling it to be inserted into similar sites of pPROBE-gfp[LAA]53. In consequence, this Probe-lux results in plasmid pPROBE-luxIR. PCR amplification of the lux operon in pPROBE-luxIR with oligonucleotides ca837F(5'-GGTATGCGGCCGCTTAATTTTTAAAGTATGGGCAATC-3') and ca837R (5'-CACTTGGATCCGTAATGACAGATAATTTTACTC-3') and then the insertion of this operon into the NotI and BamHI sites of pAC-TetInv results in the construction of the plasmid pAC-Lux Inv. This method enables a replacement of the tet promoter with the lux operon. Finally, the last plasmid that was constructed was Plasmid pAC-LuxGfp. PCR amplification was carried on the luxand gfp genes of plasmid pPROBE-luxIR through the oligonucleotides ca837F and ca803R (5'-GCAACGGTCTCGAATTCCCTTAGCTCCTGAAAATCTCGC-3'). NotI and BsaI, served as the digestion enzymes to enable its insertion into the NotIand EcoRI sites of plasmid pAC-TetInv.

The function of this plasmid was to constitutively express the invasion protein. Anchored in the outer membrane, invasin is a long rigid protein that extends 18 nm from the bacterial cell surface. This protein enables the internalization of bacteria into mammalian cells namely tumour cells including gepithelial, hepatocarcinoma, and osteosarcomaline, without any additional known adhesion molecules. However, bacterial internalization is only possible in particular cell density, hypoxia, and inducible inputs. In fact, engineered bacteri are non-invasive under conditions of cell-density and normal aerobic growth,. Sensors are only activated above a critical cell density or in a hypoxic environment resulting in the synthesis of invasin fromY. Pseudotuberculosis followed by the invasion of HeLa cells.

Anderson, J. C., Clarke, E. J., Arkin, A. P., & Voigt, C. A. (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of molecular biology, 355(4), 619-627.