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 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

Due to the major research side of our project involving actinomycetes not E.coli this part was cloned into an appropriate actinomycete-specific intergrative plasmids PMS82 and pAU3-45. This was to allow the reporter to be utilised in screening for antimycin producing actinomycetes. The plasmids containing the reporter sequence (Bba_K1041002)was transformed into specialised E.colistrains: 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 aspected.

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 Fig 2,3,4 showed the DNA is of good quality as strong 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 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 98% matches with the expected DNA 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

User Reviews

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