3. E. coli

So once you have your plasmid of interest transformed into E. coli, you will need to filter out the E.coli that have your plasmid from those that don't. This involves a passive filtering called selection and an active filtering called screening.


Selection:

Selection is the passive elimination of colonies that don't have your plasmid of interest either by positive or negative selection.

Positive selection is when your cells gain a resistance trait from your construct. So, if you have cells that have uptaken your construct, they will have resistance to the antibiotic, and will be able to grow where others cannot.

So, let's say that here's your couple of cells and some have your plasmid, you want only those on your plate, so you have a resistance on your plasmid for the "pink" antibiotic and you plate all cells on this antibiotic.

 Overnight, your colonies of interest (those with the construct) will survive and grow while those without the plasmid will die off.


So positive selection is not limited to antibiotics as many people think.
While it is more commonly used for agrobacterium, auxotroph cell-lines also allow for a positive selection. So auxotrophic cell lines are ones that have been mutated so that they are unable to create an essential compound that it needs to live, this means you can transfer a plasmid that confers the ability to create that compound, and only those cells that have taken up the plasmid will live.

    E.g., let's say you have a line of cells that are unable to make the amino acid thymidine, well, if you    transfer your construct which has the transcriptional unit for thymidine in the backbone, then only those with your construct will be able to grow and multiply. The plasmid helps fill in the missing gaps of the metabolism of the cell line.


Negative selection is when your cell lines lose a toxic trait when they uptake your plasmid. So a certain cloning technique called gateway cloning takes advantage of the ccdb suicide gene. Your insert will replace this portion of the vector so that your cells with your insert will survive and those that have the ccdb will self-cull.





Screening:

Screening is a more active filtering on your end. It can either be visual (eg. the old school blue vs white screening) or based on bench work like PCR or RE. 

PCR-based screening can be done before you make liquid cultures and can therefore give results earlier on in the process - just make sure you have a primer on the inside and outside of the insert, otherwise, you may just end up amplifying some orphaned DNA on the plate that isn't in a colony.

You can also wait till after you grow up a liquid culture of your colony for more telling results from RE screening. After extracting your plasmid (aka mini-prepping) from your liquid culture, choose REs and test them in silico before testing them on your plasmid. Make sure you get the anticipated results before moving forward.

The last step of screening is to make sure you sequence your plasmid/insert to ensure you have no SNPs. You can do this anywhere that does Sanger sequencing, but here are the two most commonly used by our lab: plasmidsaurus for whole plasmid sequencing and Azenta for insert sequencing.

    Plasmidsaurus is around $15 while Azenta is around $5 (plus shipping). Plasmidsaurus usually gets results back to us in a day or two and Azenta takes around 2 days.





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