Part:BBa_K2942707:Design

CMV promoter, riboswitch,nitroreductase and eGFP

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 901
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 901
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 901
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 901
  Illegal AgeI site found at 1456
  
• 1000
  COMPATIBLE WITH RFC[1000]

CMV promoter + Riboswitch + Nitroreductase + eGFP

Nitroreductase is an enzyme that can catalyze the reduction of nitro aromatic compounds to aromatic amines. In our project we optimized the nitroreductase that we carried out human codon optimization on the original nitro reductase and added flag tag for labeling, which Increases the expression of protein as well as detection . The CMV promoter(BBa_I712004)+ Riboswitches(BBa_k2942701)+Nitroreductase(BBa_k2942703)+eGFP(BBa_I914891)was linked into the expression vector by restriction sites EcoRI and PstI, and the correct construction of this recombinant plasmid was confirmed by PCR identification(Fig.1), sequencing of the recombinant plasmid (Fig.2)and western blot(Fig.3). The activity of the nitroreductase which can turn the prodrug to the chemicals killing cells are assessed by measuring the survival rate of the cells (Fig.4).

Primers for homologous recombination: PSBA3-F AACTGCAGTCCGGCAAAAAAGGGCAAG PSBA3-R CGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGG CMV-F TAAGGATGATTTCTGGAATTCGTGATGCGGTTTTGGCAGT CMVR-RIBO-R CGGTGACAGCTTTGTTTGTTTAGCTCTGCTTATATAAACCTCC RIBOF AAACAAACAAAGCTGTCACCGGATGTGC RIBO-GFP-R CTCCTCGCCCTTGCTCACCATGTTTTTATTTTTCTTTTTGC NR-F ATGGACATCATCTCCGTGGCCCTGAAGC FLAG-R CTCCTCGCCCTTGCTCACCATCTTATCGTCATCGTCTTTGTA GFPF ATGGTGAGCAAGGGCGAGGAGCTGT GFPR CCTTTTTTGCCGGACTGCAGCTTGTACAGCTCGTCCATG

Primers for PCR: F TTCGCTAAGGATGATTTCTGGAATTC R CCTTTTTTGCCGGACTGCAGCTTGTACAGCTCGTCCATG

Primers for sequencing: F TTCGCTAAGGATGATTTCTGGAATTC R CTTGTACAGCTCGTCCATG

Fig.1 PCR result. We extracted plasmids from the bacteria solution which shows correct sequencing result, and 1%agarose gel electrophoresis was performed to validate the PCR production of the fusion segment that has inserted between the restriction sites EcoRI and PstI. The primers for PCR are as above.

Fig.2 Sequencing result of the recombinant plasmid. The primers for sequencing are as above. You can click here to download the sequence files.

Fig.3 Transfection cells observed under a fluorescence microscope. 293T cells were seeded 7.5×105 per well in the 6 well plate, and transfection was performed following the protocol of Invitrogen Lipofectamine™ 3000 Transfection Reagent when cells grow to 70-80% of the wells. We added 4mM theophylline (solved in PBS) into the experimental group according to the Bell’s article [1] while equivoluminal PBS to control group 4h after transfection. the Fluorescence was observed 24h after the transfection.

Fig.4 Western blot result. 2×SDS was used to lyse cells when fluorescence 80% of cells can be een with fluorescence.12% SDS-PAGE was used for electrophoresis of denatured protein, and then Millipore Immobilon PVDF membrance (0.45um) was used for following immunoraction(using SIGMA Monoclonal ANTI-FLAG®BioM2−Biotin, Clone M2) and chemiluminescence reaction.

Fig.5 Survival rate of cells. 293T cells were seeded 104 per well in the 96 well plate, and CB1954(10 Um, see the reaction principle in Fig.6) and theophylline(4 mM) were added 8h after transfection..The survival rate of cells was examined using MTT Cell Proliferation and Cytotoxicity Assay Kit.

Fig.6 The principle of reaction between CB1954 and nitroreductase. Elsie M. Williams, et al. Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility. Biochem J 15 October 2015; 471 (2): 131–153.

[1] Bell, C.L., et al., Control of alphavirus-based gene expression using engineered riboswitches. Virology, 2015. 483: p. 302-311.

In conclusion, this result well confirmed that nitroreductase transformant certainly turn the prodrug to the effective chemicals.

Design Notes

we optimized the nitroreductase that we carried out human codon optimization on the original nitro reductase and added flag tag for labeling, which Increases the expression of protein as well as detection .

Source

The CMV promoter+ Riboswitches+Nitroreductase+eGFP was linked into the expression vector by restriction sites EcoRI and PstI, and among these fragments we use three restrict enzyme as following: AflIII,KpnI AND PacI.