When you insert DNA into a bacteria, why does it make your protein?

[LogicX]

So we are learning about vectors and how you can insert vectors into a plasmid and then that plasmid in a bacteria will make lots of copies of the protein that your sequence codes for.

I don't understand how inserting the DNA that codes for a protein into a bacteria gives you just the protein you want. Now, instead of just your DNA that codes the protein you want, that segment is buried within another sequence of bacterial DNA. So wouldn't a new protein be made? How is the same protein made from a sequence that is now just the middle of a larger sequence of different DNA?

Do you have to bind the vector to a stop codon on both sides or something so only that fragment codes for the amino acids that make the protein?

Also, one more question that may be relevant. Does a strand of DNA code for many different proteins on one strand? It must, or our 23 chromosomes would only make 23 proteins. So maybe the answer to my question is that the protein can be made from this small segment on the plasmid while the other parts of the sequence make different proteins, and the portions of the sequence do not form one giant protein.

 

[Mike H]

When one transforms the plasmid containing the sequence of interest into bacteria, the entire plasmid is taken up. Given that in addition to your sequence of interest, there is a replication origin, some sort of regulatory mechanism for inducing transcription, a gene that confers antibiotic resistance, and possibly other genes, it would not be suitable for that plasmid to be getting incorporated into the bacterial genome.

The protein is only produced after one has induced (over)expression, typically via chemical means. The textbook example is IPTG and the lac operon. The inducer is added, the genes on the plasmid are transcribed, and the resulting RNAs are translated.

As I believe should be clear, there can be many genes on the same strand. A typical plasmid might code for your protein of interest, an enzyme that chews up a particular antibiotic, and additional RNA polymerase so as to enhance expression.

 

[Ygggdrasil]

In general, genes have the following structure:

promoter --- Transcription start site ---Translation start site --- coding region --- translation stop site --- transcription stop site

The promoter contains DNA sequences that bind to the transcription factors that turn the gene on or off. The transcription start site is surrounded by sequences that binds to proteins that position the RNA polymerase to begin mRNA synthesis at the transcription start site. RNA polymerase then proceeds to read through the gene, synthesizing RNA until it encounters the transcription stop site, which tells RNA polymerase to fall off of the DNA and release the mRNA for translation by the ribosome. The mRNA will not contain the promoter region of the gene, but it will contain a 5' untranslated region, the translation start site, the coding region, the translation stop site, and a 3' untranslated region. The untranslated regions do not code for protein, but instead play regulatory roles to control the stability and degradation of the mRNA.

On the mRNA, the translation start site is surrounded by sequences that position the ribosome to begin protein synthesis at that location. It is always an ATG codon that codes for the amino acid methionine. The coding region contains the DNA sequences that are read by the ribosome and tell the ribosome the amino acid sequence of the protein to be synthesized. The translation stop site consists of one of three codons that tell the ribosome to stop protein synthesis.

When we insert a gene into a vector for protein expression, we generally cut out only the coding region of the gene (including the translation start and stop sites), and paste this into a vector that already contains a promoter, transcription start site, and transcription start site. Different vectors have been designed to have different promoter sequences, which can give you control over how much protein you produce. Some promoter sequences tell the bacterium to make a lot of protein, some tell the bacterium to make only a small amount of protein, and some tell the bacterium to make the protein only under certain conditions (for example, only if a certain chemical is present).

You are correct that if we were to insert our protein coding sequence into a random position of the vector, we would not expect the resulting plasmid to allow the bacterium to express our protein. The plasmid is capable of allowing expression, only because we insert our protein coding sequence into the correct location in the vector.

Because genes are demarcated by DNA sequences that specify the start and end of mRNA synthesis, we can have many genes on a chromosome.

 

[aroc91]

Also, one more question that may be relevant. Does a strand of DNA code for many different proteins on one strand? It must, or our 23 chromosomes would only make 23 proteins.

This begs the question: How did you get far enough to learn about transformation and still find it necessary to clarify the concept of many genes existing on a chromosome? No offense meant, I'm honestly confused.

 

[LogicX]

This begs the question: How did you get far enough to learn about transformation and still find it necessary to clarify the concept of many genes existing on a chromosome? No offense meant, I'm honestly confused.

Yeah... this is my first biochem course and sometimes when I make threads here random, wrong ideas pop into my mind and I just write them down. I promise I'm not that dumb :D Although, funny thing, this is my first biochem course but they never really tell you general concepts, they just want to dive into specifics.

Thanks for the help everyone.