why sometimes big or small colonies? - (Jun/28/2009 )
eldon on Jul 7 2009, 06:01 PM said:
So explain a bacterial lawn of growth. Nutrients on an LB plate are in excess and can support a lawn of bacteria for up to a month or longer.
Very easily!
A lawn is made up of a huge number of small colonies (due to the large number of cell plated). The small colonies are all suffering from nutrient starvation and the toxicity of waste products.
If, as you claim "nutrients on an LB plate are in excess....." the bacterial lawn would continue to grow for " a month or longer" and fill the dish and eventually flood over the top of the dish!
The lawn MAY survive for a some time in the dish, but the bugs are not growing actively...
I agree with klinmed -- "not growing" is not the same as "dead". I believe the large vs small colony phenotype you're seeing is simply a matter of varying cell density on the plate, and thus is correctable by proper dilution.
You can test this for yourself easily enough -- carefully pick a single small colony and streak it to a plate in the usual manner, such that a quandrant of the plate will have well-isolated single colonies.
If the size of the colonies is a function of plasmid copy number or other such genotypic influence, the well-isolated colonies in this area of the plate will be "small". If it's just a matter of how crowded the neighborhood was on the original plate, these colonies will be "large".
My money's on "large"...
HomeBrew on Jul 8 2009, 06:38 AM said:
You can test this for yourself easily enough -- carefully pick a single small colony and streak it to a plate in the usual manner, such that a quandrant of the plate will have well-isolated single colonies.
If the size of the colonies is a function of plasmid copy number or other such genotypic influence, the well-isolated colonies in this area of the plate will be "small". If it's just a matter of how crowded the neighborhood was on the original plate, these colonies will be "large".
My money's on "large"...
my money is on large too.
One more thing to add..............recombination and toxicity by gene expression
For example, if you transform a retro or lenti vector encoded hTERT into competent cells, you will see all sizes of colonies.
For example, if you transform a lentiviral vector into TOP10, the bigger colonies are recombinants in most of cases.
For example, if you transform pcDNA3.1 expressing a transcription factor, the bacteria cannot take it since the gene expression (leak expression from CMV promoter) makes the bacteria sick.
So, size matters, bigger colony might not be better sometimes.
Functional Screens on Jul 8 2009, 05:34 PM said:
For example, if you transform a retro or lenti vector encoded hTERT into competent cells, you will see all sizes of colonies.
For example, if you transform a lentiviral vector into TOP10, the bigger colonies are recombinants in most of cases.
For example, if you transform pcDNA3.1 expressing a transcription factor, the bacteria cannot take it since the gene expression (leak expression from CMV promoter) makes the bacteria sick.
So, size matters, bigger colony might not be better sometimes.
Totally agree with Functional Screens. At the far extreme, a leaky vector and a toxic expressed product = no colonies at all containing intact plasmid.
IF your colonies are well spaced, and they are of different sizes, ALWAYS pick a mix of small and large for subsequent screening.
Eldon (earlier) has suggested that different colony size on a single plate is dependent on copy number. This is nonsensical! After a short period of growth, all bugs containing a particular plasmid will contain roughly the same number of plasmid copies. How many depends on the origin of replication. For example with a ColE1 origin ca 400/ cell while with p15A ca 10.
jiajia1987 on Jun 29 2009, 12:37 PM said:
Instead of tooth picks what about the pipette tips?
DNA on Oct 27 2009, 01:39 AM said:
They work, of course -- but compare the cost of a box of tips to a box of toothpicks...