Difference between revisions of "Part:BBa K216008:Experience"
(→User Reviews) |
(→User Reviews) |
||
| Line 16: | Line 16: | ||
The right hand Biobrick site is apparently damaged. SpeI is unable to cut the part in its current form, although it can be cut with EcoRI, XbaI, and PstI. | The right hand Biobrick site is apparently damaged. SpeI is unable to cut the part in its current form, although it can be cut with EcoRI, XbaI, and PstI. | ||
| − | |||
Sequencing reveals a mutation at the SpeI site. | Sequencing reveals a mutation at the SpeI site. | ||
| + | |||
| + | '''Mutation in LuxB''' | ||
| + | |||
| + | There is a mutation in the luxB gene which causes an Ala->Arg replacement. It appears that this does not affect functionality (see below). | ||
| + | |||
| + | '''Confirmation of activity''' | ||
Colonies grown up overnight and exposed to filter paper on the lid of the petri dish with 20 ul of n-decanal glow when exposed for 30 minutes in the Alpha-Inotech Fluorchem imager. | Colonies grown up overnight and exposed to filter paper on the lid of the petri dish with 20 ul of n-decanal glow when exposed for 30 minutes in the Alpha-Inotech Fluorchem imager. | ||
Revision as of 19:01, 31 March 2010
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K216008
Comparison to other luminescent reporter systems: the quantum yield of bacterial luciferase is much lower than that of firefly or Renilla luciferase, meaning that the luminescence is much fainter. However, the substrate, n-decanal, is extremely cheap compared to the D-luciferin and coelenterazine required by these other enzymes, and if luxCDE are provided, the organism can produce its own substrate (We are in the process of preparing a luxCDE BioBrick to accompany this one; the activity of the artificial luxCDE operon has been confirmed in a non-BioBrick format). Thus bacterial luciferase is a good choice for environmental applications, where supplying luciferin or coelenterazine would not be feasible.
User Reviews
Initial experience: Edinburgh iGEM 2009
To confirm that this BioBrick works, we added it to the PyeaR promoter (BBa_K216005), which is inducible by nitrate and nitrite, and plated it on a plate with about 20 mg of solid sodium nitrate added at one edge. The following morning, colonies were present all over the plate apart from within 1 cm or so of the region where the sodium nitrate had been added (apparently it had inhibited growth in this region). N-decanal (5 microlitres) was added to the plate, which was then sealed with parafilm and returned to the incubator for 30 minutes to allow aldehyde to diffuse into the cells. The plate was then examined in a dark room. Glowing colonies were clearly visible, indicating that active LuxAB was being produced. Liquid subcultures were made in LB with ampicillin, and after overnight growth (with sodium nitrate to induce), decanal (0.5 microlitres per ml of culture) was added. Luminescence was clearly visible in the vials (see picture below). For information on further tests, see the Experience page for BBa_K216016.
Problems cutting at the SpeI site
The right hand Biobrick site is apparently damaged. SpeI is unable to cut the part in its current form, although it can be cut with EcoRI, XbaI, and PstI.
Sequencing reveals a mutation at the SpeI site.
Mutation in LuxB
There is a mutation in the luxB gene which causes an Ala->Arg replacement. It appears that this does not affect functionality (see below).
Confirmation of activity
Colonies grown up overnight and exposed to filter paper on the lid of the petri dish with 20 ul of n-decanal glow when exposed for 30 minutes in the Alpha-Inotech Fluorchem imager.
UNIQ74ff0c70682ee659-partinfo-00000000-QINU
UNIQ74ff0c70682ee659-partinfo-00000001-QINU