Difference between revisions of "Part:BBa K1094000"
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The part was cloned into expression vector pJAM1786 (via pDONR207) using the Gateway system by Invitrogen. The plasmid was transformed into ''E. coli'' cells. Colony PCR was carried out and colonies were inoculated into liquid culture. The following day the colonies failed to show significantly more fluorescence than three control cultures. Excitation was done with 485 nm and emission peak was measured at 500 nm.  
 
The part was cloned into expression vector pJAM1786 (via pDONR207) using the Gateway system by Invitrogen. The plasmid was transformed into ''E. coli'' cells. Colony PCR was carried out and colonies were inoculated into liquid culture. The following day the colonies failed to show significantly more fluorescence than three control cultures. Excitation was done with 485 nm and emission peak was measured at 500 nm.  
  
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This was unexpected since we previously showed significant fluorescence from a very similar sequence. Due to two internal PstI sites the part was not BioBrick compatible. After removing the restriction sites fluorescence was lost even though no change in amino acid sequence was made.  
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This was unexpected since we previously showed significant fluorescence from a very similar sequence. Due to two internal PstI sites that sequence was not BioBrick compatible. After removing the restriction sites fluorescence was lost even though no change in amino acid sequence was made. Therefore, we expect the lack of fluorescence to be caused cloning/transformation technical issues and not the sequence. Due to time limitation we are not able to support this statement.  
  
 
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Revision as of 07:44, 3 October 2013

Enhanced flavin-binding fluorescent protein (eFbFP)

The reporter eFbFP is a fluorescent protein that uses flavin mono nucleotide (FMN) as a cofacter. The reporter is suitable for use in anaerobic conditions.

The part was cloned into expression vector pJAM1786 (via pDONR207) using the Gateway system by Invitrogen. The plasmid was transformed into E. coli cells. Colony PCR was carried out and colonies were inoculated into liquid culture. The following day the colonies failed to show significantly more fluorescence than three control cultures. Excitation was done with 485 nm and emission peak was measured at 500 nm.

This was unexpected since we previously showed significant fluorescence from a very similar sequence. Due to two internal PstI sites that sequence was not BioBrick compatible. After removing the restriction sites fluorescence was lost even though no change in amino acid sequence was made. Therefore, we expect the lack of fluorescence to be caused cloning/transformation technical issues and not the sequence. Due to time limitation we are not able to support this statement.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 376