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Re: help with report on Plasmid Properties

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Re: help with report on Plasmid Properties

Postby lilgenius » Thu Oct 25, 2007 8:50 pm

Hi,

I am writing a lab report on plasmid properties. For the Introduction section, we have to discuss these questions:

a) What a plasmid is and how plasmids are used in genetic cloning?

b) How bacteria can acquire antibiotics resistance(through process of transformation of the bacteria).

c) How geneticists use this acquisition of antibiotics resistance to isolate the transformed bacteria.

d) How the lac Z gene gives the bacteria the ability to breakdown X-gal. What happens to the bacterial colony that allow geneticist to determine which bacterial colonies have a lac Z gene in working order and why that matters to a geneticist doing gene cloning.

I would really appreciate with somebody could help with these questions or give some answers.
:(
:x
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Postby mith » Thu Oct 25, 2007 9:09 pm

You have no business writing a lab report on plasmids if you don't know what they are
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Postby Biologista » Fri Oct 26, 2007 6:19 pm

Plasmids are the small circular DNA in bacteria. we can use them in molecular biology to insert DNA sequences (genes) into other cells or to knock out some genes and see how this affects the cell's functionning.

bacteria acquire antibiotics resistance by mutating the DNA sequence that is responsible for making it sensitive to this antibiotic. 9you needed more info?)

as for the lac Z gene.
Lac O is an operon (group of genes that work together, only found in prokaryotes) that conveys three types of genes:
1) Lac Z , or z gene
2) y gene, Lac y
3) a gene or Lac a

Lac Z encodes for beta-galactosidase that is a lactase: cleaves lactose (the sugar).
Lac y encodes for Galactoside permease that promotes the entry of lactose into the cell.
lac a encodes for transacetylase, that adds an acetyl group to the lactose molecule making it more soluble and facilitating its entry into the cytoplasm.

regulation of Lac O:
-presence of glucose represses lac O (negative control): in the presence of glucose, a repressor protein is released, it is able to bind to Lac O DNA sequence only if lactose is absent. The repressor prevents the binding of RNA polymerase and hence prevents the transcription of the Lac O. 9not total inhibition of Lac O when both glucose and lactose are present)
-abscence of glucose activates Lac O (positive control): cAMP is low when glucose levels are high. therefore as cAMP rises , Lac O is activated : cAMP acts by binding to a protein called CRP to form (cAMP,CRP) complex that would bind to the operator domain of Lac o preventing the binding of Lac O repressor and so , Lac O is activated and transcribed, beta galactosidase is secreted and breaks down lactose . now X-gal has a similar configuration to lactose and can be broken down by beta galactosidase, only difference is that X-gal is more soluble and does not need galactoside permease to enter int the cell.
we put lactose in a medium where we are culturing our bacteria along with only a low percentage of glucose (that would be used by the bacteria before 14 hours have gone), and we test for color change to see if the bacteria broke down lactose or not. if it did not break it down this suggests two hypothesis, either the plasmid contains no lac y and so the lactose molecules could not be ingested into the cytoplasm, and/or the lac z is not present and lactose cannot be fermented. so we repeat the same procedure using X-gal instead of lactose in order to see if this bacteria has lac Z (if X-gal were used - and this is indicated by a shift in color of the agar, i believe it turns blue if X-gal was used) coding for beta galactosidase or not .
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Postby MrMistery » Sat Oct 27, 2007 7:03 pm

actually i have heard of plasmids in EK cells too
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
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Postby Biologista » Sun Oct 28, 2007 11:22 am

it is usually found in bacteria and sometimes in eukaryotes like Saccharomyces cerevisiae; but mainly it is characteristic of bacteria.
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