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 an Nde1 site after their promoter sequence, enabling a restriction digest to be performed. The RFP coding gene was excised from BBa_K1041000 and ligated downstream of the AntG promoter of BBa_K1041001 to create this 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 received showed the DNA was good quality as strong chromatographic peaks were produced throughout analysis of the sample (Figs 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 received back from the sequencing company was aligned using BLAST with the expected DNA sequence (Figs 5,6).The sequencing with both the forward and reverse primers had over 96% matches. 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 K1041002 sequencing data aligned with the expected DNA sequence

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