1. You repeat DRT experiment with a Gram-positive rod and this is the result. Yo
ID: 61105 • Letter: 1
Question
1. You repeat DRT experiment with a Gram-positive rod and this is the result. Your water bath maintained a constant temperature; your dilution and plating technique is perfect. All the colony morphologies match that ove your original culture. What would cause this result? (1 point)
2. LacI and LacZ are proteins encoded by genes, and there can be mutated forms of them. The place where LacI binds, lacO, is also a DNA element, and is subject to mutation. In the table below, I’ve listed a number of potential mutations. I want you to tell me after growing each mutant in a minimal medium with equal parts glucose and lactose, both sugars at 0.2% do you think you’d see blue in a ?-galactosidase assay? (2 points)
Blue or no color change?
Mutation:
A LacI mutated so that it no longer recongizes lacO.
A LacI that no longer binds lactose
A LacZ that no longer cleaves lactose
A lacO mutated so LacI no longer recognizes it
A one base frame shift mutation after the first 9 bases of lacI
SecUnds 10000 1000 CFU/ml 100 10 0 100 200 300 400 500 600 700 Time in SecondsExplanation / Answer
1)-
Cannot understand the DRT experiment- if you can elaborate the experiment, it will be helpful.
2)-
If Beta-galactosidase is produced (due to the transcription of the lacZ gene in the lac operon), then in presence of suitable substrate, blue colour can be obtained in a beta-galactosidase assay.
In absence of lactose, the LacI proteins binds to lacO & inhibits the lac operon. But in presence of glucose & lactose, both at 0.2%, lac operon is activated as allolactose binds to the LacI & prevents it from binding to lacO (lac operator), thereby enabling the transcription of lac genes - lacZ, lacY & lacA genes. Hence Beta-galactosidase is produced due to transcription of lacZ gene & eventual translation of lacZ mRNA.
i- LacI mutated so that it no longer recognizes lacO- blue (lac operon is always switched on- hence poduction of Beta-galactosidase).
ii- LacI that no longer binds lactose- no blue colour (lac operon is always repressed as allolactose cannot bind & inactivate LacI).
iii- LacZ that no longer cleaves lactose- no blue colour. ( blue colur is formed when Beta-galactosidase cleaves subtrate X-galactose, an analog of lactose to produce a blue coloured indole product).
iv- LacO mutated so LacI no longer recognizes it- blue colour (lac operon is always switched on- since LacI cannot inhibit lac operon as it cannot bind to mutated lacO).
v- one base frame shift mutation after the first 9 bases of lacI- blue colour (probably) ( assuming the frame-shift mutation distorts the structure of LacI such that it cannot bind to lacO & thereby inhibit the lac operon).
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