Difference between revisions of "Part:BBa K1041002"

(→‎Characterisation)
 
(11 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K1041002 short</partinfo>
 
<partinfo>BBa_K1041002 short</partinfo>
  
−
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.
+
Team NRP-UEA_Norwich 2013 created this part using biobricks <partinfo>BBa_K1041000</partinfo> and <partinfo>BBa_K1041001</partinfo>. 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.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 18: Line 18:
 
==Characterisation==
 
==Characterisation==
 
Characterisation of this biobrick involved sequencing, restriction digests and BLAST analysis.
 
Characterisation of this biobrick involved sequencing, restriction digests and BLAST analysis.
 +
 +
===Restriction Digest===
 +
Part Bba_K1041002 was digested with enzyme NdeI and compared to uncut DNA (''Fig 1''). The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.
 +
[[image:BIORAD 2013-09-18 16hr 02min 2.JPG|thumb|left|Fig 1: Lane 1 contains and Bba_K1041002 cut with NdeI and lane 2 uncut Bba_K1041002.]]
 +
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
  
 
===Sequencing===
 
===Sequencing===
−
The biobrick was sent off to a company for sequencing.
+
The biobrick was sent off to a company for sequencing and the data received showed the DNA was good quality as strong chromatographic peaks were produced throughout analysis of the sample (''Figs 2,3,4'').
  
−
[[image:3a vf2 0-540.JPG|thumb|left|K1041000 sequencing data part 1]]
+
[[image:K1041002 1.JPG|thumb|left|Fig 2:K1041002 sequencing data part 1]]
−
[[image:3a vf2 440-860.JPG|thumb|left|K1041000 sequencing data part 2 ]]
+
[[image:K1041002 2.JPG|thumb|left|Fig 3:K1041002 sequencing data part 2]]
−
[[image:3a vf2 860-.JPG|thumb|left|K1041000 sequencing data part 3]]
+
[[image:K1041002 3.JPG|thumb|left|Fig 4:K1041002 sequencing data part 3]]
  
 
<br>
 
<br>
Line 35: Line 58:
 
<br>
 
<br>
 
<br>
 
<br>
 +
 
===BLAST Analysis===
 
===BLAST Analysis===
−
The data we recieved back from the sequencing company was aligned using BLAST with the expected DNA sequence ''fig.2''
+
The data we received back from the sequencing company was aligned using BLAST with the expected DNA sequence (''Figs 5,6'').The sequencing with both the forward and reverse primers had over 96% matches. 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.
 +
[[image:6 Fwd.JPG|thumb|left|Fig 5: Forward primer K1041002 sequencing data aligned with the expected DNA sequence]]
 +
[[image:6 Rev.JPG|thumb|left|Fig 6: Reverse primer K1041002 sequencing data aligned with the expected DNA sequence]]

Latest revision as of 22:13, 4 October 2013

AntG Promoter + RFP Coding Device

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.

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 683
    Illegal AgeI site found at 795
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterisation

Characterisation of this biobrick involved sequencing, restriction digests and BLAST analysis.

Restriction Digest

Part Bba_K1041002 was digested with enzyme NdeI and compared to uncut DNA (Fig 1). The enzyme digest shows the NdeI restriction has been conserved after ligation of fragments from parts Bba_J04450 and Bba_K1041001.

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

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 received back from the sequencing company was aligned using BLAST with the expected DNA sequence (Figs 5,6).The sequencing with both the forward and reverse primers had over 96% matches. 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 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