Difference between revisions of "Part:BBa K1041000"

(→‎Restriction Digests)
(→‎Restriction Digests)
Line 30: Line 30:
  
 
[[image:K1041000 digest 2.JPG|thumb|left|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.]]
 
[[image:K1041000 digest 2.JPG|thumb|left|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.]]
−
[[image:K1041000 digest.JPG|thumb|left|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.]]
+
[[image:K1041000 digest.JPG|thumb|centre|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.]]
−
<br>
+
−
<br>
+
−
<br>
+
−
<br>
+
−
<br>
+
−
<br>
+
  
 
===Sequencing===
 
===Sequencing===

Revision as of 13:47, 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

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

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