Q&A about molecular cloning, plasmids and vectors #Everyone is a Writer

Synthesis killed the cloning star♭♭

I personally think DNA synthesis will eventually kill molecular cloning which is sad because It's a satisfying process to do and my strongest skill. Who has not felt like a cloning star after finding the correct insert in your Macrogen trace file? It is most probable that we the ones working now with it are the last of the Mohicans of Gibson Assembly and who knows how many more generations of scientists will continue to find molecular cloning methods relevant. One may think that as long as people use plasmids for anything, there will be cloning but we are near a time (or is it already here?) when synthesis advances enough to make it cheaper to have your gene inserted by just writing. Will molecular cloning be removed from the skill set required by everyday biologists? Probably. But imagine not having to do Gibson, then transformation, then purification to find that you made a mistake and just order an engineered plasmid with the exact resistance and insert gene you wanted, no need for assembly, or selection. It would be so nice to not have to waste time with those initial cloning steps and for academics it means that you can get more done in the same amount of time.

What is molecular cloning?

Gene cloning for me is any technique that multiplies a gene. The ingredients are always a fragment of DNA with some gene and a circular DNA molecule called vector. First open the vector with restriction enzymes or in a pcr reaction with a very powerful polymerase that makes no mistakes, then mix both of them in a tube and you get your recombinant DNA molecule. For the next step you need to mix it with the host cell which will engulf your vector and multiply it against their will. The gene carried by the recombinant molecule is now said to be cloned.

Source: Brown, T. A. 2013. Gene Cloning and DNA Analysis: An Introduction. John Wiley & Sons.

What's the difference between a plasmid and a vector?

A vector is not always a plasmid. It can also be something else like the chromosome of a virus or any molecule that can carry a gene into a host cell which is the requisite to be a vector. A plasmid is just the most popular vector.

What are the necessary components of a plasmid?

What a plasmid cannot lack is a replication origin to replicate inside the host cell and a selectable marker to tell which cells have engulfed it. Also a polylinker which is a cluster of cloning sites artificially made that allow restriction enzymes to cut and open the plasmid in those locations to receive the insert. Nowadays you can open your plasmid in any location if using Gibson assembly.

Source: Youtube

What are the facts that determine plasmid copy number?

The time will come when you encounter protocols that ask you if your plasmid is low copy number or high. What is it and why is it important? The reasons for being a multicopy plasmid (those with guarantee that you will have at least 20 copies per cell) are still a mystery but a hint that you can be working with a low copy number plasmid is that yours is one of the larger ones. In nature you can find plasmids ranging from about 1.0 kb for the smallest to over 250 kb for the largest plasmids. Only some of them are useful, 10kb is a nice number to work with and design your molecular cloning strategy. Copy number can also be increased using a protein synthesis inhibitor such as chloramphenicol that will mess with chromosomal replication but your plasmid will be replicating like crazy.

How many DNA cloning methods have been developed?

There is traditional restriction enzyme cloning and then there are the new methods which possess one big advantage: they are significantly cheaper than traditional cloning, in terms of both materials and time. You could say that there are many DNA cloning methods as ideas. Gibson Assembly from NEB is one of them, In-Fusion by Takara-Bio (formerly Clontech) is another one. Behind each of them there is a great idea. Both of them seems to be the future and rely on the use of an exonuclease to chunk the DNA fragments to make seamless assemblies of as many fragments as you wish and don't require linearization of the plasmids using restriction enzymes due to the use of potent polymerases that made virtually no error in copying your plasmid and opening at the same time.

What vector can be used to clone a very large DNA fragment?

Plasmids are not sufficient to clone large fragments that is why if you would want to clone a big human gene of 100kb you will need to search other category of vectors which are based on yeast or bacterial chromosomes, yeast chromosomes called YACs can hold up 600kb easily.

What are the commonly used vectors for gene expression in prokaryotic and eukaryotic systems?

Almost all nowadays plasmids have something left of one of the most famous plasmids: the pBR322. ‘p’ indicates that this is indeed a plasmid. ‘BR’ identifies the laboratory in which the vector was originally constructed. For protein expression in E. coli it is common to use the pBAD, pET, pHAT series which are derived from the beloved pBR322. For Eukaryotic systems like yeast the pYES2 vector is very common.

What are the free tools for plasmid design and plasmid mapping?

The best way is to take a piece of paper and draw your plasmid map, then store it in a black carpet with the label “top secret” and stick it in a dusty bookshelf in between your labs collection of old Sigma-Aldrich catalogs. If you don't have any of that in your lab you can use online tools like Benchling or the online tool in the GenScript webpage.