Difference between revisions of "Part:BBa K1604022"

Revision as of 15:41, 10 September 2015

araC-pBAD + β-carotene + J23100 + blh

This biobrick is obtained by the assembly of two parts K1604020 and K1604021. The composite part contains the five genes necessary for the retinal synthesis. The first part contains ctrEIBY under araC-pBAD, these four gene are responsive of beta-carotene production starting from FPP, a colurless precurse presents in E.coli. The second part contains blh under costitutive promoter. It encodes for the beta-carotene15-15'dioxygenase that catalizes the cleavege of a single molecule of beta-carotene into two molecules of retinal.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 9
Illegal NheI site found at 32
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2041
Illegal BamHI site found at 4082
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 247
Illegal NgoMIV site found at 3618
Illegal NgoMIV site found at 3748
Illegal AgeI site found at 1876
Illegal AgeI site found at 2833
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1858

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1604022 short</partinfo>
@@ Line 7: / Line 6: @@
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+<div style="text-align:center">[[]]</div>
+<p style="width:600px; margin-left:150px; margin-bottom:60px;
+text-align:justify "><b>FIGURE 1. Loss of &#946;-carotene. NEB&#946; cells were cotransformed with BBa_K1604021 (in puC57) and BBa_ K1604020 (aracpBad-&#946; carotene). Positive control was BBa_K1604020. The cells were grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. BBa_K1604022 (&#946; carotene) induced (A); cotrasformation of BBa_K1604021 and BBa_ K1604020 (B),  with 5 mM arabinose (C), and with 5 mM arabinose, 5 uM FeSO4 and 10 mM of ascorbate (D); empty cells (E). Expression of blh causes the loss of the typical orange colored pellet of &#946;  carotene expressing cells. </b> </p>
+<div style="text-align:center">[[]]</div>
+<p style="width:600px; margin-left:150px; margin-bottom:60px;
+text-align:justify "><b>FIGURE 2. Extraction of &#946; carotene and retinal. NEB&#946; cells were cotransformed with BBa_K1604021 (in puC57) and BBa_ K1604020 (aracpBad-&#946; carotene). BBa_K1604020 only was used for positive control. The cells were grown in 100 mL of LB and induced as described in figure 1. After 24 hours the cells were span down and the supernatant was discarded. The pellets were incubated with 2.5 mL of acetone for 10 min at 50C. Afterward they were centrifuged to recover the extracted pigments. The samples were diluted 1:7 in acetone and the spectra were taken with UV-VIS Agilent .... equipped with a deuterium and tungsten lamps. The spectra were acquired between 300 and 800 nm and blanked with acetone. Panel A extraction in acetone of carotenoids and retinoids. Panel B UV VIS spectra: BBa_K1604020 (&#946; carotene) with arabinose Fe and ascorbate (purple), BBa_K1604020 (&#946; carotene) + BBa_K1604021 (blh) with Fe and ascorbate. </b> </p>
+Our data show that there is a loss of &#946; carotene when the cells express blh. The UV_Vis analysis confirmed the loss of &#946; carotene (452 nm), but did not evidenced the presence of retinal (373 nm).  For a more detailed characterization please check BBa_K1604022.
 <!-- -->