Part:BBa_K1041000:Experience

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Team NRP-UEA_Norwich 2013

Team NRP-UEA_Norwich 2013 added an NdeI restriction site at the start of the RFP coding region of BBa_J04450 using mutagenesis. This was to allow either the promoter region or RFP gene to be excised and exchanged for a different promoter or gene and provide restriction sites for further cloning such as part Bba_K1041002.

Characterisation of this biobrick involved comparisons with the original Bba_J04450 biobrick by performing transformations, restriction digests and BLAST analysis. We also had our biobrick sequenced.

Transformation

Parts BBa_K1041000 and BBa_J04450 were transformed into Alpha-Select competent E.Coli cells and plated onto LB Agar plates which contained the antibiotic ampicillin. Both plates have colonies that appear red under natural light Fig.1. Therefore the addition of a NdeI site has not effected the ability for the RFP gene to be transcribed.

Fig 1: Plates containing (left) BBa_K1041000 and (right) BBa_J04450 visualized under non-UV lightbox

Restriction Digests

Parts Bba_K1041000 and Bba_J04450 were analysed by restriction digests and run on agarose gels. Both biobricks were cut with NdeI, Fig 2 and PvuII Fig 3.

Fig 2:Analysis of Restriction enzyme digest with NdeI. Lanes 1 and 2 contain uncut Bba_K1041000 and Bba_K1041000 digested with NdeI. Lanes 3 and 4 contain uncut Bba_J04450 and Bba_J04450 digested with NdeI respectively.

Fig 3:Analysis of Restriction enzyme digest with PvuII. Lanes 2 and 3 contain uncut BBa_J04450 and BBa_J04450 digested with PvuII respectively. Lane 5 contains Bba_K1041000 with PvuII.

Sequencing

The biobrick was sent off to a company for sequencing, Fig 3,4,5.The data we recieved back showed the DNA is of good quality.

Fig. 3:K1041000 sequencing data part 1

Fig. 4:K1041000 sequencing data part 2

Fig. 5:K1041000 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 6,7

Fig. 6: Forward primer K1041000 sequencing data aligned with the expected DNA sequence

Fig. 7: Reverse primer K1041000 sequencing data aligned with expected DNA sequence

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