Difference between revisions of "Part:BBa K1041001"
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<partinfo>BBa_K1041001 short</partinfo>
 
<partinfo>BBa_K1041001 short</partinfo>
  
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This part contains the promoter sequence AntG, where the unique sigma factor AntA binds to activate transcription of the neomycin resistance gene. There is a Nde1 resticition site between the promoter and gene to allow either to be removed and exchanged for a different promoter or gene.
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Team NRP-UEA_Norwich 2013 designed this part to contain the promoter sequence AntG, where the unique sigma factor AntA binds to activate transcription of the neomycin resistance gene. There is a Nde1 resticition site between the promoter and gene to allow either to be removed and exchanged for a different promoter or gene. This facilitated cloning of the biobrick <partinfo>Bba_K1041002</partinfo>
  
 
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<partinfo>BBa_K1041001 parameters</partinfo>
 
<partinfo>BBa_K1041001 parameters</partinfo>
 
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==Characterisation==
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Characterisation of this biobrick involved sequencing, restriction digests and BLAST analysis.
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===Restriction Digests===
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Part Bba_K1041001 was cut with enzyme NdeI and compared to the uncut DNA (''Fig 1''). The enzyme digest shows that there is an NdeI restriction site as expected.
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[[image:BIORAD 2013-09-18 16hr 02min.JPG|thumb|left|Fig 1: Lane 1 contains uncut Bba_K1041001 and Lane 2 Bba_K1041001 cut with NdeI.]]
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===Sequencing===
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The biobrick was sent off to a company for sequencing and the data we received back (''Figs 2,3,4'') showed the DNA was good quality as analysis of the sample produced strong chromatographic peaks throughout the analysis of the sample.
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[[image:K1041001 1.JPG|thumb|left|Fig 2:K1041001 sequencing data part 1]]
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[[image:K1041001 2.JPG|thumb|left|Fig 3:K1041001 sequencing data part 2]]
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[[image:K1041001 3.JPG|thumb|left|Fig 4:K1041001 sequencing data part 3]]
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===BLAST Analysis===
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The data we received back from the sequencing company was aligned using BLAST with the expected DNA sequence (''Fig 5,6''). The sequencing with both the forward and reverse primers had over 98% matches with the expected DNA sequence. Deviations from the expected sequence were at the end of the useful sequencing data and combination of both sets of data confirmed a complete match with the expected sequence. 
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[[image:1 Fwd.JPG|thumb|left|Fig 5: Forward primer K1041001 sequencing data aligned with the expected DNA sequence]]
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[[image:1 Rev.JPG|thumb|left|Fig 6: Reverse primer K1041001 sequencing data aligned with the expected DNA sequence]]

Latest revision as of 21:59, 4 October 2013

Neomycin Resistance Coding Device + AntG promoter

Team NRP-UEA_Norwich 2013 designed this part to contain the promoter sequence AntG, where the unique sigma factor AntA binds to activate transcription of the neomycin resistance gene. There is a Nde1 resticition site between the promoter and gene to allow either to be removed and exchanged for a different promoter or gene. This facilitated cloning of the biobrick BBa_K1041002

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 757
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 606
    Illegal SapI.rc site found at 816


Characterisation

Characterisation of this biobrick involved sequencing, restriction digests and BLAST analysis.

Restriction Digests

Part Bba_K1041001 was cut with enzyme NdeI and compared to the uncut DNA (Fig 1). The enzyme digest shows that there is an NdeI restriction site as expected.

Fig 1: Lane 1 contains uncut Bba_K1041001 and Lane 2 Bba_K1041001 cut with NdeI.


















Sequencing

The biobrick was sent off to a company for sequencing and the data we received back (Figs 2,3,4) showed the DNA was good quality as analysis of the sample produced strong chromatographic peaks throughout the analysis of the sample.


Fig 2:K1041001 sequencing data part 1
Fig 3:K1041001 sequencing data part 2
Fig 4:K1041001 sequencing data part 3










BLAST Analysis

The data we received back from the sequencing company was aligned using BLAST with the expected DNA sequence (Fig 5,6). The sequencing with both the forward and reverse primers had over 98% matches with the expected DNA sequence. Deviations from the expected sequence were at the end of the useful sequencing data and combination of both sets of data confirmed a complete match with the expected sequence.

Fig 5: Forward primer K1041001 sequencing data aligned with the expected DNA sequence
Fig 6: Reverse primer K1041001 sequencing data aligned with the expected DNA sequence