Part:BBa_K4727000
A. baumannii & K. pneumoniae standard backbone
This plasmid backbone was developed by Wang et al [1], it is provided of two ORIs: the first one a pMB1/pUC high copy number ORI for E. coli, the second one is capable o maintaining the plasmid both in Acinetobacter baumannii and Klebsiella pneumoniae. The plasmid brings KanR as a selection marker
This backbone has been made standard RFC[10] compatible and provided with the RFC[10] prefix and suffix. This, would allow to easily interchange genetic parts and express them in two non model organisms as Acinetobacter baumannii and Klebsiella pneumoniae. This part, complemented by the contribution work made by our team on parts <partname>BBa_J23119</partname>, BBa_R0040, BBa_B0010 and BBa_B0012 helps bringing synthetic biology a step further in non model organisms.
Usage and Biology
This part it thought to be used to express synthtic constructs in non model organisms as Acinetobacter baumannii and Klebsiella pneumoniae. It has been successfully used to express a reporter expression cassette in these two species, see image above. We conducted a fluorescence emission analysis employing the Thermofisher Scientific Varioskan Lux plate reader. This assessment focused on the emission of a reporter gene, specifically the red fluorescent protein (RFP) with excitation at 535nm and emission at 610nm. Results are reported below.
Acinetobacter baumannii
Our experimental setup involved four colonies of A. baumannii, each harboring the RFP reporter gene within a cassette integrated into our newly developed backbone. To establish a comparative benchmark, we contrasted these expression levels with those of a standard E. coli TOP10 strain expressing the RFP gene under the control of both the relatively weak promoter BBa_J23101 and the strong promoter BBa_R0040.
Among the selected colonies, namely 1.2A, 1.2B, 1.1A, and 2.2A, a trio comprising 1.2A, 1.2B, and 1.1A exhibited analogous rates of reporter gene expression. This expression level was higher than the signal produced by BBa_J23101 promoter driving RFP; yet, it remained notably lower than the signal produced by a E.coli TOP10 strain bearing a pTet promoter driving RFP in the high copy plasmid pSB1A2 (iGEM part BBa_I13521) . Conversely, colony 2.2A distinctly displayed heightened expression, comparable to the one showed in E. coli by the I13521 cassette.
Upon subjecting the expression cassettes to Sanger sequencing, we determined that the diminished fluorescence emission stems from a mutation within the RFP coding sequence. This mutation, rather than the promoter itself, leads to a significant reduction in emission intensity due to an amino acid substitution.
Klebsiella pneumoniae
The experiment was carried out in a similar fashion as described above: two colonies of K. pneumoniae were electroporated, with part BBa_K4727000 expressing a gene reporter, namely RFP, under the regulation of BBa_J23119 promoter. In parallel, the same wild type bacteria was used as a reference, together with our golden standards: E. coli expressing RFP under the control of pTet and BBa_J23101.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found at 3664
Illegal suffix found at 3721 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3664
Illegal NheI site found at 969
Illegal NheI site found at 3692
Illegal NheI site found at 3715
Illegal SpeI site found at 3722
Illegal PstI site found at 3736
Illegal NotI site found at 3670
Illegal NotI site found at 3729 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3664
Illegal BglII site found at 1162
Illegal XhoI site found at 1775
Illegal XhoI site found at 3780 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 3664
Illegal suffix found at 3722 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 3664
Plasmid lacks a suffix.
Illegal XbaI site found at 3679
Illegal SpeI site found at 3722
Illegal PstI site found at 3736 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal SapI site found at 4008
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