Part:BBa_K1041001:Experience

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Team NRP-UEA_Norwich 2013

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 an Nde1 resticition site between the promoter and gene to allow either to be removed and exchanged for a different promoter or gene, which facilitated cloning of the biobrick BBa_K1041002

Due to the major research side of our project involving actinomycetes and not E. coli, this part was also cloned into the appropriate actinomycete-specific integrative plasmids PMS82 and pAU3-45. The resulting plasmids allowed the neomycin reporter gene to be utilised in screening for antimycin producing actinomycetes. The resulting plasmids that contained the reporter sequence from Bba_K1041002 were transformed into specialised E. coli strains: ET12567 and pUZ8002. These cells were then conjugated with spore stocks of actinomycetes cultivated from soil samples to transfer the active biosensor in the plasmid.

Characterisation

This involved sequencing, restriction digests and BLAST analysis.

Restriction Digest

Part Bba_K1041001 was cut with enzyme NdeI and compared to the uncut DNA (Fig 1). Fig 1 shows 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 is of good quality as strong chromatographic peaks were produced throughout 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

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