Difference between revisions of "Part:BBa K2942708:Design"
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<partinfo>BBa_K2942708 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2942708 SequenceAndFeatures</partinfo> | ||
| + | ===CMV promoter + Riboswitch + CPG2 + eGFP=== | ||
| + | Carboxypeptidase is a kind of peptidase that specifically degrades and releases free amino acids from the C end of the peptide chain. And the ligand we use in our project is G2 type which could cleave C-terminal Glu under N-acetyl-L-aspartyl-L-glutamate structure. CPG2 is a zinc-dependent dimeric protein with no mammalian analogue [1], thus in our project we carried out human codon optimization on the original CPG2 and added flag tag for labeling to increase protein expression and make detection easier. CPG2 rapidly hydrolyzes extracellular MTX to its non-toxic metabolites, called 2, 4-diamino-N10-methypteroic acid and glutamic acid [2], so it can be used for the treatment of elevated plasma concentrations of MTX. The principle of this reaction shows in the picture as follows. | ||
| + | The CMV promoter(BBa_I712004)+ Riboswitches(BBa_K2942701)+CPG2 (BBa_k2942704)+eGFP(BBa_I914891) was linked into the vector pSB1A3 by restriction sites EcoRI and PstI , and the correct construction of this recombinant plasmid was confirmed by PCR identification(Fig.1) and sequencing of the recombinant plasmid (Fig.2).The expression of nitroreductase can be indirectly refleted by observing the fluorescence(Fig.3) and directly seen using western blot(Fig.4). 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.5). | ||
| + | |||
| + | Primers for homologous recombination: | ||
| + | PSBA3-F AACTGCAGTCCGGCAAAAAAGGGCAAG | ||
| + | PSBA3-R CGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGG | ||
| + | CMV-F TAAGGATGATTTCTGGAATTCGTGATGCGGTTTTGGCAGT | ||
| + | CMVR-RIBO-R CGGTGACAGCTTTGTTTGTTTAGCTCTGCTTATATAAACCTCC | ||
| + | RIBOF AAACAAACAAAGCTGTCACCGGATGTGC | ||
| + | RIBO-CPG-R GCACAAGGCCGCCAGCTCCATGTTTTTATTTTTCTTTTTGC | ||
| + | CPG-F ATGGAGCTGGCGGCCTTGTGCC | ||
| + | FLAG-R CTCCTCGCCCTTGCTCACCATCTTATCGTCATCGTCTTTGTA | ||
| + | GFPF ATGGTGAGCAAGGGCGAGGAGCTGT | ||
| + | GFPR CCTTTTTTGCCGGACTGCAGCTTGTACAGCTCGTCCATG | ||
| + | |||
| + | Primers for PCR: | ||
| + | F TTCGCTAAGGATGATTTCTGGAATTC | ||
| + | R CCTTTTTTGCCGGACTGCAGCTTGTACAGCTCGTCCATG | ||
| + | |||
| + | Primers for sequencing: | ||
| + | F TTCGCTAAGGATGATTTCTGGAATTC | ||
| + | R CTTGTACAGCTCGTCCATG | ||
| + | |||
| + | |||
| + | [[file:7081.png]] | ||
| + | |||
| + | |||
| + | 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. | ||
| + | |||
| + | |||
| + | [[file:7082.png|720px]] | ||
| + | |||
| + | 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. | ||
| + | |||
| + | In conclusion, this result well confirmed that nitroreductase transformant certainly turn the prodrug to the effective chemicals. | ||
| + | |||
| + | [1] Bell, C.L., et al., Control of alphavirus-based gene expression using engineered riboswitches. Virology, 2015. 483: p. 302-311. | ||
===Design Notes=== | ===Design Notes=== | ||
Revision as of 12:56, 20 October 2019
CMV promoter+ Riboswitches+ CPG2+eGFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2105
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1411
Illegal NgoMIV site found at 1609
Illegal NgoMIV site found at 2038
Illegal AgeI site found at 2119 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 961
CMV promoter + Riboswitch + CPG2 + eGFP
Carboxypeptidase is a kind of peptidase that specifically degrades and releases free amino acids from the C end of the peptide chain. And the ligand we use in our project is G2 type which could cleave C-terminal Glu under N-acetyl-L-aspartyl-L-glutamate structure. CPG2 is a zinc-dependent dimeric protein with no mammalian analogue [1], thus in our project we carried out human codon optimization on the original CPG2 and added flag tag for labeling to increase protein expression and make detection easier. CPG2 rapidly hydrolyzes extracellular MTX to its non-toxic metabolites, called 2, 4-diamino-N10-methypteroic acid and glutamic acid [2], so it can be used for the treatment of elevated plasma concentrations of MTX. The principle of this reaction shows in the picture as follows. The CMV promoter(BBa_I712004)+ Riboswitches(BBa_K2942701)+CPG2 (BBa_k2942704)+eGFP(BBa_I914891) was linked into the vector pSB1A3 by restriction sites EcoRI and PstI , and the correct construction of this recombinant plasmid was confirmed by PCR identification(Fig.1) and sequencing of the recombinant plasmid (Fig.2).The expression of nitroreductase can be indirectly refleted by observing the fluorescence(Fig.3) and directly seen using western blot(Fig.4). 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.5).
Primers for homologous recombination: PSBA3-F AACTGCAGTCCGGCAAAAAAGGGCAAG PSBA3-R CGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGG CMV-F TAAGGATGATTTCTGGAATTCGTGATGCGGTTTTGGCAGT CMVR-RIBO-R CGGTGACAGCTTTGTTTGTTTAGCTCTGCTTATATAAACCTCC RIBOF AAACAAACAAAGCTGTCACCGGATGTGC RIBO-CPG-R GCACAAGGCCGCCAGCTCCATGTTTTTATTTTTCTTTTTGC CPG-F ATGGAGCTGGCGGCCTTGTGCC 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.
In conclusion, this result well confirmed that nitroreductase transformant certainly turn the prodrug to the effective chemicals.
[1] Bell, C.L., et al., Control of alphavirus-based gene expression using engineered riboswitches. Virology, 2015. 483: p. 302-311.
Design Notes
The CMV promoter+ Riboswitches+Nitroreductase+eGFP was linked into the expression vector by restriction sites EcoRI and PstI ,and among these fragments we use the following restrict enzyme AflIII,KpnI and PacI in order to link them.We also carried out human codon optimization on the original CPG2 and added flag tag for labeling to increase protein expression and make detection easier.
Source
The promoter and eGFP were cloned from other plasmids,and CPG2 and riboswitch were artificially synthesized.