Part:BBa_K1041002:Experience

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Team NRP-UEA_Norwich 2013

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.

Due to the major research side of our project involving actinomycetes and not E. coli this part was also cloned into an appropriate actinomycete-specific integrative plasmid PMS82. This was to allow the reporter to be utilised in screening for antimycin producing actinomycetes. The plasmid containing the reporter sequence from 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.

One further experiment was designed to activate the RFP gene by transforming BBa_K1041002 into an E. coli BL21 strain along with the plasmid pET28AntA which synthesises the sigma factor AntA upon induction of T7 RNA polymerase by addition of IPTG. It was hoped that with both plasmids in the same cell, the AntA sigma factor would be produced and activate transcription of the RFP gene by binding to the promoter sequence antGP. This would mean that the cells could be spread onto agar plates containing Chloramphenicol and the colonies would appear red under natural light. The experiment was performed, but unfortunately the colonies did not appear red. To check that both plasmids were in the E. coli BL21 cells, DNA was purified from liquid cultures and analysed by restriction enzyme digests and agarose gel electrophoresis (Fig 1). The figure shows that both plasmids were contained within the E. coli BL21 cells as expected. Thus, further experiments are required to optimise this system and to discover why the sigma factor is not activating synthesis of the red fluorescence protein.

Fig 1: Analysis of 'E. coli BL21 cells containing pETAntA and BBa_K1041002 plasmids compared to the DNA of the plasmids alone digested with XbaI, NdeI and no enzyme.

Characterisation

This involved sequencing, restriction digests and BLAST analysis.

Restriction Digest

Part Bba_K1041002 was digested with enzyme NdeI and compared to uncut DNA (Fig 2). The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.

Fig 2: Lane 1 contains 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 we received back (Figs 3,4,5) showed the DNA was good quality as strong chromatographic peaks were produced throughout analysis of the sample.

Fig 3: K1041002 sequencing data part 1

Fig 4: K1041002 sequencing data part 2

Fig 5: 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 6,7). The sequencing with both the forward and reverse primers had over 96% matches to the expected 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 6: Forward primer K1041002 sequencing data aligned with the expected DNA sequence

Fig 7: Reverse primer K1041002 sequencing data aligned with the expected DNA sequence

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