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in vivo cloning, transforming cells

Hi there
So I was wondering if someone would help me out with A2 content

In the book it says that 'If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
If a vector is a bacteriophage, it will infect the host bacterium by injecting its DNA into it.'

Can someone explain that in more detail please?
Have a good day. :biggrin:

Reply 1

HarisMalik98

Bacteria don't tend to show the natural ability to uptake plasmids (transformation). Transformation is a complex, energy-requiring process so typically bacteria won't do this naturally.

In order to counteract this, in the lab, Bacteria need to made 'competent' in order to uptake plasmids.

This can be done by a few different ways, e.g. a common way is treatment with Calcium Chloride (CaCl2) followed by heat-shock. The Ca2+ ions bind the negatively-charged plasmid DNA backbone and the negatively-charged core groups on the bacterial LPS proteins (in the outer cell membrane) and bring the plasmid to the cell surface. Heat-Shock is when the solution is heated to ~42C which creates pores in the cell membrane and allows the plasmid to enter.

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During phage infection (transduction), the phage facilitates the entry of the genetic material into the bacteria itself when it infects the bacterial cell. The bacteria do not control this process.

In essence, Bacteria usually need to undergo special treatment to uptake plasmids themselves. Whereas during phage infection, the phage itself introduces the genetic material - so the bacteria 'don't need to do anything'.

Hope this helps.

(edited 3 years ago)

Reply 2

username5266058

Original post by harismalik98

bacteria don't tend to show the natural ability to uptake plasmids (transformation). Transformation is a complex, energy-requiring process so typically bacteria won't do this naturally.

In order to counteract this, in the lab, bacteria need to made 'competent' in order to uptake plasmids.

This can be done by a few different ways, e.g. A common way is treatment with calcium chloride (cacl2) followed by heat-shock. The ca2+ ions bind the negatively-charged plasmid dna backbone and the negatively-charged core groups on the bacterial lps proteins (in the outer cell membrane) and bring the plasmid to the cell surface. Heat-shock is when the solution is heated to ~42c which creates pores in the cell membrane and allows the plasmid to enter.

--------------------------------------------------------------------------------------------------------------------------------------------------------------------

during phage infection (transduction), the phage facilitates the entry of the genetic material into the bacteria itself when it infects the bacterial cell. The bacteria do not control this process.

In essence, bacteria usually need to undergo special treatment to uptake plasmids themselves. Whereas during phage infection, the phage itself introduces the genetic material - so the bacteria 'don't need to do anything'.

Hope this helps.

thank you so much!!