-signal activates its kinase activity → phosphorylate the response regulator to change its conformation so that it can actively bind to DNA (promoter region)

-triggers a change in gene expression to either promote production of proteins needed to respond to sensation or even turn off expression of proteins that be making it respond negatively to changes in environment

-response regulator is physically attached to the sensor (membrane bound)

entire system is constrained to the membrane
system has to navigate around the membrane and/or DNA target has to shift to meet the response regulator

Two-Component System

-2 domains are separate proteins

-sensor detects the signal and interacts with second protein (response regulator)

-response regulator is free-floating in cytoplasm

able to actively find and bind to its target
elicits a faster response because it’s not constrained

Knock-Out Mutants and Complements

Laboratory Plasmids

origin of replication (Ori): allows plasmid to replicate independently of chromosome
often 2 selection/selectable markers: allows researchers to selectively grow the cells that have taken up the plasmid
multiple cloning sites (MCS): regions where researchers can insert a target gene and/or promoter region
expression plasmids
- produces a protein of interest; protein of interest doesn’t need to be bacterial in nature
recombinant plasmids
- plasmid that has another species protein cloned in it
- normally have MCS in front of existing promoter
reporter plasmids
- to not express a gene of interest but determine whether a gene of interest is being expressed
- promoter of gene of interest is cloned in front of reporter protein where a read-out can be easily measured
- ex: GFP derived from jellyfish or lacZ (beta-galactosidase) enzyme

Knock-Out Mutants

-laboratory derived strains where researchers have deleted a gene of interest to see what happens to the phenotype being studied

ex: researchers interested in seeing if fliC (involved with flagellar biosynthesis, swimming motility) → researchers knock-out (remove) fliC → look for absence or inhibition of swimming

-if gene is essential for that phenotype → expect phenotype to be attenuated or absent in mutant compared to wild type

Complements

-deleting a gene from the chromosome can have unforeseen effects on various elements

-polar effects

side effects that don’t contribute to phenotype loss seen in a knock-out mutant

-researchers complement mutant by reintroducing the original gene on an expression plasmid into the cell

-goal is to see if phenotype can be restored by expressing the original gene in isolation

if phenotype restored → confidently attribute that phenotype loss was due to that single gene deletion
if phenotype not restored → polar effects contributing to phenotype loss that may not necessarily be because of the target gene removed

Knowt