Part:BBa_K5082010

Usage and Biology

Plasmids are double stranded circular DNA molecules, which carry non-essential genes, found in bacterial cells [1]. Plasmids are multifunction, easy to edit, and can be amplified quickly by bacterial cellular machinery [2]. These advantages have made plasmids a popular tool in genetic research.

pMIR is a mammalian expression vector of the Luciferase reporter gene with a CMV promoter [3]. The pMIR plasmid can be amplified by DH5α bacterial cells. Meanwhile, it carries an ampicillin resistance gene for selection. The plasmid map for pMIR is shown in Figure 1.

                       
                             Figure 1. pMIR plasmid map.

Design

The above characteristics have made pMIR an ideal plasmid for our experiment. In our experiment, we fused an HSU structure downstream the Luciferase gene to visualize G3BP1 concentration and thereby diagnosed gastric cancer. Meanwhile, we also transfected empty plasmid backbones and plasmids with MUT regions downstream GFP into stomach cell lines to serve as control groups.

Characterization

The pMIR plasmid carries an ampicillin resistance gene. Therefore, we cultured bacterial cells containing the plasmid on solid medium with ampicillin. As shown in Figure 2, the bacteria were successfully cultured, proving that the plasmids have been successfully transformed.

                    
                          Figure 2. pMIR bacterial cell culture.

Reference

[1] Carattoli, Alessandra. “Plasmids in Gram Negatives: Molecular Typing of Resistance Plasmids.” International Journal of Medical Microbiology, vol. 301, no. 8, Dec. 2011, pp. 654–658

[2] Patron, N.J. Synthetic Biology and Gene Cloning. Elsevier EBooks, Elsevier BV, 1 Jan. 2017, pp. 112–117

[3] “Addgene: PCMV-GFP.” Www.addgene.org, www.addgene.org/vector-database/3582/.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 7332
  Illegal SpeI site found at 5344
  Illegal PstI site found at 2047
  Illegal PstI site found at 5601
  Illegal PstI site found at 7344
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7320
  Illegal SpeI site found at 5344
  Illegal PstI site found at 2047
  Illegal PstI site found at 5601
  Illegal PstI site found at 7344
  Illegal NotI site found at 92
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5090
  Illegal BamHI site found at 2792
  Illegal XhoI site found at 7326
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 7332
  Illegal SpeI site found at 5344
  Illegal PstI site found at 2047
  Illegal PstI site found at 5601
  Illegal PstI site found at 7344
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 7332
  Illegal SpeI site found at 5344
  Illegal PstI site found at 2047
  Illegal PstI site found at 5601
  Illegal PstI site found at 7344
  Illegal NgoMIV site found at 2498
  Illegal NgoMIV site found at 5708
  Illegal NgoMIV site found at 7052
  Illegal NgoMIV site found at 7073
  Illegal AgeI site found at 1822
  Illegal AgeI site found at 3926
  Illegal AgeI site found at 5209
  Illegal AgeI site found at 6776
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3752
  Illegal BsaI.rc site found at 1435
  Illegal SapI.rc site found at 2347
  Illegal SapI.rc site found at 2557
  Illegal SapI.rc site found at 6958