Difference between revisions of "Part:BBa K911004:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | 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. | how you used this part and how it worked out. | ||
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| + | 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. We strongly recommend that you do not attempt to assemble it in psB1C3, as it will kill your cells. | ||
| + | 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. We suspect that the repeated terminator may facilitate recombination, and another team might 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. | ||
| + | Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent) | ||
| + | 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. | ||
===Applications of BBa_K911004=== | ===Applications of BBa_K911004=== | ||
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| + | 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. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 13:24, 22 September 2012
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.
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. We strongly recommend that you do not attempt to assemble it in psB1C3, as it will kill your cells. 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. We suspect that the repeated terminator may facilitate recombination, and another team might 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. Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent) 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.
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.
User Reviews
UNIQd082236f7157bad7-partinfo-00000000-QINU UNIQd082236f7157bad7-partinfo-00000001-QINU