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 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.

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

One further experiment was designed to activate the RFP gene by transforming BBa_K1041002 into a BL21 E.Coli strain along with the plasmid pET28AntA which synthesises the sigma factor AntA. 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 Chlorophenicol and the colonies would appear red under natural light. Unfortunately the colonies did not appear red. We then decided to confirm that both plasmids were in the BL21 cells by analysing the DNA by restriction enzyme digests and performing agarose gel electrophoresis, Fig 1.

Fig 1:Analysis of 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 enzymes XbaI and 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 and Bba_K1041002 cut with XbaI and NdeI and lane 2 uncut Bba_K1041002.

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 good quality as strong peaks were produced throughout analysis of the sample.

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 5,6The 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

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