Difference between revisions of "Part:BBa K216008:Experience"

Latest revision as of 22:32, 27 October 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.

Edinburgh 2010 Experience and Improvements

We attempted to use this BioBrick this year from the stock we had of last years submitted DNA. As it turns out, the luxAB submitted was not the working version. Both Edinburgh and Mexico-UNAM-Genomics attempted several times to transform E.coli with this construct and visualise luminescence but never successfully. We eventually produced a working version of this gene which, when transformed into E.coli, causes it to visibly glow in the presence of decanal. We have resubmitted this to the registry this year with a fused lacZ promoter (BBa_K322140).

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. See the sequencing analysis data for this and for the mutation below.

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.

UNIQbbae99318502d94b-partinfo-00000000-QINU UNIQbbae99318502d94b-partinfo-00000001-QINU

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 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
+===Edinburgh 2010 Experience and Improvements===
+We attempted to use this BioBrick this year from the stock we had of last years submitted DNA. As it turns out, the luxAB submitted was not the working version. Both Edinburgh and Mexico-UNAM-Genomics attempted several times to transform ''E.coli'' with this construct and visualise luminescence but never successfully. We eventually produced a working version of this gene which, when transformed into ''E.coli'', causes it to visibly glow in the presence of decanal. We have resubmitted this to the registry this year with a fused lacZ promoter (BBa_K322140).
 ===Applications of BBa_K216008===
@@ Line 11: / Line 15: @@
 '''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. For information on further tests, see the Experience page for BBa_K216016.
+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.  See the sequencing analysis data for this and for the mutation below.
+'''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.
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