Difference between revisions of "Part:BBa K3038001:Experience"

(→‎Applications of BBa_K3038001)
(→‎Applications of BBa_K3038001)
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Characterization of the ADR constructs:
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PCR amplification of the PCR products
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T--Poitiers--PCR_amplification_ADR-tab3.jpg
  
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Electrophoresis photography following deposits on agarose gel 0.8% of enzymatic digestion products. The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder. Lane 1 corresponds to the marker, lane 2 to the digested N-ter,ADR lane 3 to the digested C-ter ADR and lane 4 to the digested plasmid pSB1A3.
  
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Electrophoresis gel photography following deposits of N-ter and C-ter ADR PCR products. The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is NEB 1 kb Plus DNA Ladder.
 
  
  
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Plasmid construction
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T--Poitiers--plasmid_construction_ADR-tab3.jpg
  
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Design of ADR N-ter/pSB1A3 and ADR C-ter/pSB1A3 with Geneious software. This map shows the pBAD promoter and its terminator flanking the coding sequence of the ADR protein. Also present in N-ter or C-ter are 6-His and c-myc tag. Finally, in the plasmid is present and ampicillin resistance cassette.
  
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Modelization of the ligation between ADR N-ter/pSB1A3 and ADR C-ter/pSB1A3 with Geneious software. This map shows the pBAD promoter and its terminator flanking the coding sequence of the ADR protein. Also present in N-ter or C-ter are 6-His and c-myc tag. Finally, in the plasmid is present and ampicillin resistance cassette.
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Expression of the CMYC-6HIS-ADR and ADR-CMYC-6HIS recombinant proteins
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T--Poitiers--recombinantexpression_ADR-tab3.png
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NI : Not induced I: Induced
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After sequencing, induction was performed on the thermocompetent bacteria JM109. The objective was to verify if the cloned gene leads to the production of a protein. The expected size of the ADR protein is 40 kDa. A very strong expression of the ADR protein was observed at this size when the pBAD promoter is induced with arabinose. The gene has therefore been correctly cloned into the strain and the protein is produced.
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Activity
  
 
===User Reviews===
 
===User Reviews===

Revision as of 08:51, 11 October 2019


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Please enter how you used this part and how it worked out.

Applications of BBa_K3038001

PCR amplification of the PCR products T--Poitiers--PCR_amplification_ADR-tab3.jpg

Electrophoresis photography following deposits on agarose gel 0.8% of enzymatic digestion products. The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder. Lane 1 corresponds to the marker, lane 2 to the digested N-ter,ADR lane 3 to the digested C-ter ADR and lane 4 to the digested plasmid pSB1A3.


Plasmid construction T--Poitiers--plasmid_construction_ADR-tab3.jpg

Design of ADR N-ter/pSB1A3 and ADR C-ter/pSB1A3 with Geneious software. This map shows the pBAD promoter and its terminator flanking the coding sequence of the ADR protein. Also present in N-ter or C-ter are 6-His and c-myc tag. Finally, in the plasmid is present and ampicillin resistance cassette.


Expression of the CMYC-6HIS-ADR and ADR-CMYC-6HIS recombinant proteins T--Poitiers--recombinantexpression_ADR-tab3.png

NI : Not induced I: Induced

After sequencing, induction was performed on the thermocompetent bacteria JM109. The objective was to verify if the cloned gene leads to the production of a protein. The expected size of the ADR protein is 40 kDa. A very strong expression of the ADR protein was observed at this size when the pBAD promoter is induced with arabinose. The gene has therefore been correctly cloned into the strain and the protein is produced.

Activity

User Reviews

UNIQc5b76677361ed14b-partinfo-00000000-QINU UNIQc5b76677361ed14b-partinfo-00000001-QINU