Difference between revisions of "Part:BBa K1084504"

Latest revision as of 10:53, 19 October 2013

Promoter Selector-mRFP

This part is promoter 9 (BBa_K1084009) constructed as Promoter Selector vector expresses mRFP1 (Red).

iGEM HokkaidoU Japan 2013 original promoter family is constructed from consensus promoter (BBa_K1084001) by adding mutation at -35 region. Other region is same among family members, and similar with promoter consensus sequence.

Fig. 1 Randomized promoter family sequences

Correspondence of sequence and parts number is below.

  -35        BBa_         t. e. rank
  TTGACA     K1084001     1   
  TAGGTC     K1084002     2
  CTGAAG     K1084003     6
  GGGGTG     K1084004     3
  GAGGAT     K1084005     5
  GCAATA     K1084006     7
  GGGGGG     K1084007     8
  TGTGTG     K1084008     4
  AGTGGG     K1084009     9
  TCTCGG     K1084010     10

t. e. = theoretical transcription efficyency

Promoter transcription efficiency was measured with mRFP1 (BBa_K1084401-K1084410), LacZ and Kanamycine (Km) resistance (BBa_K1084501-K1084505).

Fig. 2 mRFP1 assay result

Fig. 3 β-Galactosidase assay result

Fig. 4 Km resistance assay result

Fig. 5 Comparison of assay results and modeling data

Theoretical promoter modeling also has done (see promoter Background page in HokkaidoU 2013 wiki). According to protein, these promoters show relatively another protein activity revel. Choose your best promoter by promoter optimization kit (Promoter Selector). Plate image below is actual worked Promoter Selector (Km resistance).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal suffix found in sequence at 213
Illegal PstI site found at 1294
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 214
Illegal PstI site found at 228
Illegal PstI site found at 1294
Illegal NotI site found at 221
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal suffix found in sequence at 214
Illegal PstI site found at 1294
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 214
Illegal PstI site found at 228
Illegal PstI site found at 1294
Illegal AgeI site found at 70
Illegal AgeI site found at 1014
Illegal AgeI site found at 1126
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 73
Illegal BsaI.rc site found at 67

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1084504 short</partinfo>
-This part is promoter 9 (BBa_K1084009) constructed as POK vector expresses mRFP1 (Red).
+This part is promoter 9 (BBa_K1084009) constructed as Promoter Selector vector expresses mRFP1 (Red).
 iGEM HokkaidoU Japan 2013 original promoter family is constructed from consensus promoter (BBa_K1084001) by adding mutation at -35 region. Other region is same among family members, and similar with promoter consensus sequence.
@@ Line 36: / Line 36: @@
 Theoretical promoter modeling also has done (see promoter Background page in HokkaidoU 2013 wiki).
 According to protein, these promoters show relatively another protein activity revel.
-Choose your best promoter by promoter optimization kit (POK).
+Choose your best promoter by promoter optimization kit (Promoter Selector).
-Plate image below is actual worked POK (Km resistance).
+Plate image below is actual worked Promoter Selector (Km resistance).
 https://static.igem.org/mediawiki/parts/0/00/HokkaidoU_2013_POK_DEMO_48h_1.png