Difference between revisions of "Part:BBa K911004:Experience"

Latest revision as of 16:11, 16 November 2012

During and after assembly of this sequence, unexpected toxicity issues were observed. This necessitated its assembly in a low copy number vector. This part is submitted to the registry as is, in the suitable backbone provided by the synthesis company (with permission from HQ). The backbone sequence is submitted as BBa_K911006 for user reference. We strongly recommend that you do not attempt to assemble it in psB1C3, as it will kill your cells. It might also be advisable (if not essential) to transform this into an e.coli strain that can tolerate toxic inserts reasonably well, such as XL1 blue, and grow it at around 30 degrees C.

The toxicity of the construct causes strong selective pressure against it, and characterisation has been hampered by the tendency of cells to lose parts of the insert, although it does seem to be fairly stable across subsequent replating if it is retained on the initial transformation plate. Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent), as shown.

These patches are derived from either white or orange colonies on the original transformation plate

This image shows the constitutive luminescence of each patch. Note cosegregation of luminescence and colour

We suspect that the repeated terminator may facilitate recombination, and another team might wish to investigate whether replacing the second terminator aids stability.

The construct is not behaving entirely as expected, as the e.coli colonies are initially orange, despite mOrange being lacI-repressed. This is probably due to leakage, as the RBSes are very strong in both e.coli and bacillus, and retrospectively could have been predicted. This could be indicative of high sensitivity to promoter activity, which is by no means a bad thing!

We ran out of time before attempting to insert this into bacillus. Bacillus homology regions would need to be added, but on the upside, the much lower copy number would likely counteract the toxicity issues.

The emission spectrum of the construct before and after induction is shown below, compared to the unmodified lux operon BBa_325909. Despite earlier evidence to the contrary, there does not seem to be a wavelength shift. Perhaps this is because the wildtype luxA subunit can outcompete the modified one for binding to luxB (as the wt luxA is contranscribed with the rest of the operon). Or perhaps this is simply not as the original paper described it.

A graph comparing the emission spectrum of our construct (red and green) to wildtype luciferase (blue). No shoulder at 560nm is apparent.

In personal experience, IPTG induction increases toxicity issues significantly.

Applications of BBa_K911004

BBa_k911004 was designed to act as a quantitative reporter construct. It still has potential for this, but the toxicity/stability issues should be addressed.

The B.subtilus codon-optimised bioluminescent operon and mOrange sequences are both clearly fully functional, and could be extracted and used for other purposes.

User Reviews

UNIQ80adf9405c8d7325-partinfo-00000000-QINU UNIQ80adf9405c8d7325-partinfo-00000001-QINU

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 We ran out of time before attempting to insert this into bacillus. Bacillus homology regions would need to be added, but on the upside, the much lower copy number would likely counteract the toxicity issues.
-[[Image:Lux_colour_change_image_1_small.jpg|300px|left|thumb|The bacteria on the left are expressing normal luciferase (BBa_K325909) and those on the right are transformed with our construct.]]
+The emission spectrum of the construct before and after induction is shown below, compared to the unmodified lux operon [https://parts.igem.org/Part:BBa_K325909 BBa_325909]. Despite earlier evidence to the contrary, there does not seem to be a wavelength shift. Perhaps this is because the wildtype luxA subunit can outcompete the modified one for binding to luxB (as the wt luxA is contranscribed with the rest of the operon). Or perhaps this is simply not as the original paper described it.
-Initial tests with an orange filter indicate that the mOrange-luxA fusion may be producing an additional emission component around 560 nm. However this experiment is fairly ad-hoc and could produce misleading results due to different colony densities, and should not be taken as full confirmation. We will add a full emission spectrum when we have access to a scanning luminometer.
+[[Image:800px-Luciferases_graph.png|700px|center|thumb| A graph comparing the emission spectrum of our construct (red and green) to wildtype luciferase (blue). No shoulder at 560nm is apparent.]]
+In personal experience, IPTG induction increases toxicity issues significantly.
 ===Applications of BBa_K911004===