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How to select competent E. coli cells?

plasmid transformation competent cell

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7 replies to this topic

#1 xxcici

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Posted 01 March 2013 - 03:28 PM

Hi, i want to select an E. coli competent cell for my plasmid transformation. I find there are many options that I do not know how to select a optimal one. I want to know the difference of the competent cells like DH5a, JM109, etc. Can all E. coli cells be made to be competent for transformation? Or we have to make sure that there is no plasmid with antibiotics genes in the E. coli cells. Then only these E. coli strains can be used for making competent cells? I am confused now.

#2 bob1

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Posted 01 March 2013 - 06:27 PM

Any E. coli cells can be made competent, the particular strain you want to use is dependent on what you want to do with the cells in the end. For plasmid growth and extraction DH5alpha is probably the most commonly used strain, but if you want to express (off a plasmid) and then purify protein from the bacteria, BL21 is a commonly used strain.

For both these purposes, initially you want the bacteria to have no plasmids in them, so you don't use any selective antibiotics when growing them to make them competent. The most widely used methods for transformation (i.e. insertion of plasmids into the bacteria) are heat shock (called chemically competent) and electroporation (called electrocompetent). The easiest and cheapest is heat shock and follows the instructions detailed in Douglas Hanahan's 1983 paper "Studies on Transformation of Escherichia coli with Plasmids" J Mol Biol, 166, 557-580. An excellent variant of this is called Top10 (and ideally uses Invitrogens' Top10 strain, but works well with other strains too) and the protocol can be found here.

#3 xxcici

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Posted 02 March 2013 - 08:31 AM

Hi Bob1, thank you very much for your reply. I think naturally, bacteria have plasmids inside the cell, right. During the replication of the cell, plasmid is replicated. The role of plasmid in nature is protecting bacteria against antibiotics. Even if I do not add antibiotics in their growth medium, bacteria containing plasmids can grow. My question is how to make sure bacteria do not have plasmids before making them competent.

#4 bob1

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Posted 02 March 2013 - 12:26 PM

Sure, there may be plasmids in the bacteria before you try to make them competent, but if you choose to use a particular strain then your lab should have a stock that doesn't contain selectible plasmids. The plasmids you add should have an antibiotic resistance gene such as Ampicillin resistance (Ampr), that will allow you to select for them. To test that the bacteria don't have this selectible marker, plate a small amount out on an Amp plate, if they don't grow then you know that there is no Ampr containing plasmid in the bacteria.

#5 xxcici

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Posted 02 March 2013 - 01:13 PM

Great thanks to you again!

At the beginning, I think all naturally existing plasmids contain antibiotics resistant gene. So, now, it should be right that only engineered plasmids used in lab contain antibiotics resistant gene. For the bacteria become antibiotic resistant naturally, they also contain antibiotic resistant gene, right?

One more question pop up in my mind that for doing transformation of the competent cells, both many copies of the recombinant plasmids of interest and the plasmids without insertion can be taken up by a single bacterium at the same time, right? So, for a single colony, it may contain the recombinant plasmids and the initial vector together but the colony is resistant to antibiotics. If I extract plasmid from this colony, my plasmid will be impure. How to avoid this situation?

#6 bob1

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Posted 03 March 2013 - 12:22 AM

Great thanks to you again!

At the beginning, I think all naturally existing plasmids contain antibiotics resistant gene. So, now, it should be right that only engineered plasmids used in lab contain antibiotics resistant gene. For the bacteria become antibiotic resistant naturally, they also contain antibiotic resistant gene, right?

Some naturally ocurring plasmids also have antibiotic resistance genes, but in the lab you should only find strains that are susceptible unless they have an engineered plasmid inside.

One more question pop up in my mind that for doing transformation of the competent cells, both many copies of the recombinant plasmids of interest and the plasmids without insertion can be taken up by a single bacterium at the same time, right? So, for a single colony, it may contain the recombinant plasmids and the initial vector together but the colony is resistant to antibiotics. If I extract plasmid from this colony, my plasmid will be impure. How to avoid this situation?

Yes, in theory, but you should screen your colonies for the insert, so as to eliminate this possibility.

#7 xxcici

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Posted 06 March 2013 - 07:05 AM

Hi Bob, thank you very much!

I want to say something more for this question.
For doing cloning sequence to the plasmid, sometimes, plasmid ligate with itself. So, in the ligated product, plasmid is not pure.
Then the plasmid is transformed into the bacteria. All plasmids can make bacteria resistant to antibiotics. So, on the plate added with antibiotics, there will be three kinds of colonies: one with pure plasmid with right insertion, one with plasmids with insertion and without, one with plasmid ligated with itself.
If I randomly pick up one colony from the plate (added antibiotics) and extract the plasmid. I may pick up the second kind colony and get impure plasmid but I do not know. Even if I test the extracted plasmid by PCR, I still could not eliminate the possibility that there are plasmid without insertion mixed in the product, right?

#8 bob1

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Posted 06 March 2013 - 12:09 PM

There are controls that you should be doing for the ligation - these include vector with no insert in the presence of a ligase (i.e. to see if the vector will religate) and digested vector with and without insert and no ligase (to ensure that the digests have worked properly). If on your vector only + ligase control you get a large number of colonies then you should try again as you vector is re-ligating and this will form the majority of the colonies (you can also do blue/white selection to look for those with an insert if you have a lac operon on the plasmid). Presence of any colony on the vector without ligase means that your digests didn't work.

Yes, testing by PCR is not infallible, but it if you then take this DNA and sequence it (as you should for any plasmid to ensure that it has the correct sequence - this will definitely save you a lot of time and wasted experiments later if you didn't and then found out that something was wrong with the plasmid), the sequence readout you get will have a lot of mixed peaks as the two products (one from the insert and one from the vector), such that the sequence is unreadable. You can then discard this one.





Also tagged with one or more of these keywords: plasmid transformation, competent cell

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