Mutant microorganisms resistant to lactose killing
a technology of mutations and microorganisms, applied in the direction of transferases, biochemical equipment and processes, enzymes, etc., can solve the problems of difficult to synthesize molecules, high cost, and severely impaired lactose uptake of lactose killing negative strains
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example 3
The Use of Translational Coupling or Translational Sensors to Ensure Lactose Transporter Expression
[0131]Because a full lactose permease knock out strain would also not undergo lactose killing and the goal is to obtain a functional, active, expressed lactose permease a screen is needed to ensure lactose permease expression. To this end, sequence variants of promoters, ribozyme binding sites, Kozak sequences, codon usage and transcription terminators can be created. However, these sequence variants may lead to null-expression constructs, hence leading to lactose transporter negative mutant strains. Therefore a system needs to be designed to detect the expression of the lactose transporter, preferably by a easy to screen reporter gene such as lacZ, fluorescent proteins or antibiotic resistance genes.
[0132]Construction of a Translational Coupling System that Reinitiates Translation of the Reporter Gene to Detect Lactose Transporter Expression
[0133]Two genes can be translationally coupl...
example 4
Detection of the Expression of a Lactose Transporter Translational Coupled with a Chloramphenicol Resistance Gene
[0141]Two strains were constructed in which lactose permease was knockout out from the genome. In both strains a pSC101 plasmid containing a kanamycin resistance gene was transformed, with the difference that one of the plasmids contained a constitutively expressed lactose permease as described in Example 1 and 2, resulting in a reference strain MG1655ΔlacY pSC101_kan and the lacY_cat translational coupled strain MG1655ΔlacY pSC101_kan_lacYsynthetic cat as described in Example 4 and FIG. 5. Both strains were grown in a minimal medium as described above at different chloramphenicol concentrations (between 0 and 30 mg / l). FIGS. 8 and 9 show after 48 and 92 hours of growth that the growth of reference strain is inhibited at a lower chloramphenicol concentration than the mutant lacY_cat translational coupled strain, which makes such a system an excellent screen for lactose pe...
example 5
Screening Procedure for Lactose Permease Expressing Mutants that do not Undergo Lactose Killing
[0142]Similar to Example 2, a mixture of two strains resistant to chloramphenicol were grown, one strain that does not undergo lactose killing and translational coupled to chloramphenicol and one strain with the chloramphenicol cassette but with the natural expression system of lactose permease. Both strains were grown in the medium as described in Example 2 and mid exponential phase lactose was added as shown in Example 2. The mutant strain that does not undergo lactose killing kept on growing while the other strain, that is lactose killing sensitive, stopped growing. At the end of the exponential phase, 0.1 ml of this culture was inoculated in a second shake flask with a similar medium as described above. Again, at OD 0.8 lactose was added arresting the growth of the lactose killing sensitive strain and further enriching the mutant strain that does not undergo lactose killing. After 5 re...
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