Difference between revisions of "Part:BBa K1041000"

Revision as of 11:29, 18 September 2013

RFP Coding Device

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 781
Illegal AgeI site found at 893
1000
COMPATIBLE WITH RFC[1000]

Composite part of the following BioBricks:

LacI R0010	B0034	mRFP1 E1010	B0015

BBa_R0010: Promoter (lacI regulated)
BBa_B0034: RBS (Elowitz 1999)
BBa_E1010: Red Fluorescent Protein from Discosoma striata
BBa_B0015: Double Terminator

Characterisation

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

Sequencing

The biobrick was sent off to a company for sequencing, Fig 2,3,4.The data we recieved back showed the DNA is of good quality.

Fig. 2:K1041000 sequencing data part 1

Fig. 3:K1041000 sequencing data part 2

Fig. 4: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 5,6

Fig. 5: Forward primer K1041000 sequencing data aligned with the expected DNA sequence

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

@@ Line 22: / Line 22: @@
 ===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.
+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.
 [[Image:WP 000051.jpg|thumb|Fig 1: Plates containing (left) BBa_K1041000 and (right) BBa_J04450 visualized under non-UV lightbox]]