Part:BBa_K1041002

AntG Promoter + RFP Coding Device

Team NRP-UEA_Norwich 2013 created this part using biobricks BBa_K1041000 and BBa_K1041001. These biobricks both contain a Nde1 site after their promoter sequence, enabling a restriction digest to be performed. The RFP coding gene was excised from BBa_K1041000 and ligated in front of the AntG promoter of BBa_K1041001 to create a new biobrick.

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 AgeI site found at 683
  Illegal AgeI site found at 795
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

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

Restriction Digest

Part Bba_K1041002 was digested with enzyme NdeI and compared to uncut DNA Fig 1.The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.

Fig 1:Lane 1 contains and Bba_K1041002 cut with NdeI and lane 2 uncut Bba_K1041002.

Sequencing

The biobrick was sent off to a company for sequencing and the data recieved showed the DNA is good quality as strong peaks were produced throughout analysis of the sample. Fig 2,3,4..

Fig 2:K1041002 sequencing data part 1

Fig 3:K1041002 sequencing data part 2

Fig 4:K1041002 sequencing data part 3

BLAST Analysis

The data we recieved 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 96% matches.

Fig 5: Forward primer K1041002 sequencing data aligned with the expected DNA sequence

Fig 6: Reverse primer K1041002 sequencing data aligned with the expected DNA sequence