Genetic Engineering Basics

[Chowe]

Hello all,

I have a very basic understanding of genetic engineering, I understand how genes are extracted and transferred and expressed. My question is what is the barrier between expressing genes from one organism into another? From my understanding, the main problem in expressing the lux operon in plants is due to the genes originally being expressed in bacteria. But I have also read about bacteria genes being expressed in mammalian cells. Do all genes need to be codon optimized for any chance of being expressed? For example I have a pVIB vector, can I only express that vector in bacteria? Or can I express that vector in fruit flies as well? Lastly, what makes a plasmid backbone different from species to species that I can only express it in that specific species (like expressing a bacteria vector in a mammal cell)? Thanks

Corey

[Mega]

Well, first starting:

As you said, lux is an operon.

Bacterial DNA polymerase needs one promoter and then expresses many genes if no terminator ends it's voyage.

Eukaryotes need one promoter per coding site.

Additionally, the bacterial promoter won't give you any/very weak expression in plants and vice versa.

[Nathan McCorkle]

We like to teach/think of molecular interactions as a hand fitting
into a glove, or a key into a lock. Where bonds in a molecule are give
it a pretty defined 3D structure, and the type of atoms in that
structure help to define where the electrons are, which influence the
3D electron cloud structure. Opposites attract, meaning electrons are
attracted to lack-of-electrons (+ charge, AKA holes).

In general, eukaryotes are different enough evolutionarily some common
parts are just quite different, even though they have the same
function.

If you know cars, it would be like asking why can't you use the same
crankshaft from and Otto cycle gasoline engine in a Mazda RX-7 rotary
engine. They both burn gasoline and output rotary power to a shaft,
but internally they've evolved in a much different fashion.

Lots of smart folks are trying to figure out these differences so they
can re-engineer compatibility.

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--
-Nathan

[shreyans chordia]

A few important features and scenarios for a vector to work in another host:

1. Proper promoter seq on DNA where the host transcription machinery can bind.

2. Proper 5' untranslated region seq on the mRNA for the mRNA to be translated. (not mentioning codon frequency, rare codons)

3. Further for the vector to replicate in another host it should either have its own replication machinery or else be equipped (For ex. the origin of replication seq in the vector should be same as that in the host) to use the hosts replication machinery.  

4. Also replication alone is not enough, the vector needs to partition properly along with the daughter cells of the host such that there are no daughter cells without that vector.

5. This scenario is further complicated by the fact that other naturally occurring plasmids in the new host can out compete ur vector.

[Josiah Zayner]

Honestly, it really depends on the gene. The biggest problem that can be found is posttranslational regulation and protein folding. Otherwise there are many many many many many genes that have been expressed across species. Usually people just take the gene of interest and clone it into the expression vector of the organism of choice and off they go.

The expression vector you are using doesn't seem to use any specific viral polymerase like most bacterial expression vectors. However, bacteria use a Shine-Dalgarno ribosome binding sequence which is different from the Kozak sequence used in eukaryotes. As someone else already said, they also use operons which mostly do not exist in Eukaryotes.

I would suggest finding a book on molecular biology protocols to learn the basics.