21 replies to this topic

[carlmartin]

Hello Bioforum,

I hope I am posting in the right place.

I have some questions about the whole gene cloning affair in the bacteria host.

• I understand (theoretically) that plasmids in a host are replicated but do they always transcribe/translate (henceforth express) the encoded proteins?
  
• A cursory exam of plasmid maps show antibiotic resistance genes but do these genes have promoters if (1) is "yes"?
  
• The topic on gene expression control uses operon as example. Do these antibiotic genes get induced by anything or under any control?
  
• Can I simply clone a gene into a plasmid and get the host to show the new phenotype or do I need some sort of promoter to go with this gene (I am thinking along the line of PCR here)?

In the textbooks, the flow is always to obtain the gene of interest, clone it, and select for the plasmid bearing bacteria using the antibiotic markers or the expression of the gene. But is it that straightforward or are the details not mentioned?

I think there was this recent post about promoterless gene in here as well. And another one about two promoters. Both confuses me further.

Thank you to anyone who will patiently guide me through this.

Edited by carlmartin, 09 August 2012 - 08:41 AM.

[phage434]

Genes must be transcribed to RNA, then translated to protein to be expressed. Transcription is controlled by promoters, 5' of the coding region of the protein. There are promoters 5' of the antibiotic resistance genes in plasmids. Usually these are so-called constitutive promoters, meaning they are always on; some antibiotic resistance genes are somewhat toxic to cells, and the promoters for these can sometimes be turned on selectively in the presence of the antibiotic, but this is somewhat rare.
Common plasmids used for cloning often have a promoter 5' of the cloning site, so that when gene coding regions are inserted into the cloning site, the gene is expressed. Some of these promoters are inducible, that is, they can be turned on and off depending on the presence of specific chemicals, such as IPTG or arabinose. Hope this helps.

[ascacioc]

1) In order to transcribe a gene you need a promoter for the RNA polymerase to bind and produce mRNA (depending on the promoter, you might need also a terminator); on the mRNA you need upstream the start codon, an rbs (ribosome binding site) in order for the ribosome to bind and do the translation resulting in your protein. You have to take care that the origin or replication (to multiply the plasmid inside the bacteria), promoter and rbs are specific for bacteria if bacteri is used for expression, or yeast if you use yeast etc. It is possble that a plasmid has an origin of replication for bacteria in order for you to be able to clone it in bacteria, and another origin of replication for yeast, for you to use the plasmid for yeast expression.
2) See the answer above with the addition that if you have an antibiotic resistance you have to check for which organism its promoter is specific i.e. I have a plasmid that gives ampicilin resistance in bacteria, and gentamicin resistance when used for viral infection in insect cells. On the other hand, even though the gentamicin resistance is there on the plasmid, in bacteria, this does not give gentamicin resistance because the promoter is specific for insect cells expression.
3) See the answer before.
4) By phenotype are you refering to protein expression? Usually foreign gene expression does not give a phenotype other than protein expression and slower growth. Sometimes I have a phenotype when I am expressing proteins that binf DNA/RNA since it binds every free DNA and RNA and becomes toxic to the cell. If you are refering to protein expression as phenotype, it is not that easy as it looks on the paper. But to answer your question, you do not need to add a promoter, the vectors come with a promoter before the multiple cloning site in which you must insert your gene of interest.There are some details that people miss and then they get into troubles. For example, to give you only the basics:
a) There are cloning bacterial strains such as DH5alpha or XL1Blue which are lacking specific endonucleases/recombinases which make them suitable to retrieve DNA in larger amounts; you use these strains to transform your ligation reaction to get your final plasmid. Grow slow.
There are expression strains such as BL21 which are lacking specific proteases to keep your expressed protein intact. Transform previously obtained plasmid into these strains and use this for expression.
c) There is a variant of BL21 called Gold that has both the advantage of lacking the endonucleases/recombinases and being suitable for expression leading to the possibility of transforming the ligation reaction directly into this strain and skiping the DH5alpha/Xl1Blue step
d) If you are working with pET vectors (having T7 promoter) you must work with expression strains that have (DE3) in the name (meaning that they can express T7 RNA polymerase that recognizes this promoter)
e) There are cases in which even though you cloned everything right, your protein is not expressed or results in inclusion bodies or is straight misfolded and fragmented inside the bacteria. In these cases you must test different expression strains, different vectors with promoter of different strengths, different expression temperatures, different media, different N-terminal tags that promote folding, different fragments of protein because bigger than 50 kDa proteins in bacteria are difficult to express (some people say larger than 27 kDa)

The thing with two/multiple promoters is that there are vectors that are made in such a way that you can use them for expression in different organisms i.e. E coli and insect cells without needing to reclone the gene by having two promoters one after the other such that when in bacteria the RNA polymerase recognizes the bacterial promoter, when in insect cells, the insect cells RNA polymerase recognizes the insect cell promoter. i.e. pTriEX from Novagen,

What you have asked is an entire field of expertise. If this would be easy to explain in 1-2 pages, I wouldn't be needed in my job:) I could suggest some material for reading such as Unit 5.24 from Current Protocol in Protein Science: Strategies to Optimize Protein Expression in E. coli by DM Francis and R. Page. If you cannot access it from your lab, drop me a message with your email address and I will send it to you.

[carlmartin]

Thank you very much phage434 and ascacioc. I was reading about molecular biology to prepare ahead of my final year. I understand that I am still a long way out and I appreciate your taking the time to explain. Could not really sleep with so many question marks floating about but I am happy to put some of these away.

Just to note here: when I mentioned "phenotype", I was thinking along the line of a new "characteristics" conferred by the new gene. Maybe that was inaccurate and I got mixed up with Drosophila genetics The PET system is fascinating (first impression) but I am not looking to make so many proteins. Just mutant bacterial clones.

To recap what I learned:

• genes shown on maps have promoters even though they are not shown
• cloning sites have promoters to express an inserted gene
• origin of replication & others are specific to the type of host it
• I think T7 promoters are from the same T7 bacteriophage
• there are so many bacteria host out there

If someone could help me with a few more questions:

• What happens if the gene I put into the plasmid has a promoter? Will it cause a problem since transcription has to start twice; once at the resident promoter of the plasmid and another time at the new promoter?
• Any particular reference book (besides the internet) to point me to? Or is it all about experience?

Going forward I am sure I will have more questions and I will try to find out the answers by myself first.

[ascacioc]

To just make a quick note on the phenotype thing: If you are refering to a particular characteristic confered by the gene: if you have a type of cell (bacteria, yeast, mamalian cell line) that is a mutant of a gene (deletion mutant), you can make a rescue experiment using a plasmid containing the gene you deleted from the chromosome. This means that the protein will be expressed from the plasmid and will make the cell healthy (behaving like wild type) again. Plasmids are not only for expression of proteins for purification towards biochemical analysis.

Yes, T7 promoters come from the T7 bacteriophage. Studier, who according to me is the father of cloning, has characterized the bacteriphage many years ago and came up with the idea of pET system. He did also other wonderful things in cloning and protein expression like the autoinduction media. If you have time, you could look up what other contributions he did to the field of cloning.

Indeed, there are many bacterial hosts. What I find important to know is to know what a K strain (cloning strain) and a B strain (expression strain) are. Also, if you start using them, you could start learning the genotyping codes. A strain comes with a certain genotype i.e. mutations and insertions of genes, which make it different to other strains. Basically, at this level you need to read only 1-2 lines of genotype code and be able to tell what is the use of this strain. But this is expert level. A good webpage for this is: http://openwetware.o..._coli_genotypes But again, this is knowledge you have in your mind when you do only this for many years. Nobody will expect you to know these things (most of the PhD students around me are happy without even knowing that this wiki exists)

Your questions:
1) Good question. Never thought about it. When I have seen it this morning, I was like: why would you do this? But just for theoretical purposes (even though, I am not 100% sure about what I am saying next): I think that the gene will be transcribed from both promoters i.e. once by RNA polymerase binding to one promoter and another time by binding to the other promoter. On the other hand, it will be crowded in this area and this might lead to slowing down or to shorter mRNA fragments because I imagine that if the first RNA polymerase binds on the upstream promoter and there is also another RNA polymerase downstream, the the first one stumbles accross the second one and cannot continue would wait a while until it cannot move anymore and then detach. Anyhow, we do not do that
2) http://www.amazon.co...=pd_sim_sbs_b_1 This the the bible of molecular cloning. However, nobody buys this for their own (check the price). Each good lab has a copy. I learn usually from internet, like wikis. The companies that sell the strains/vectors have also good material e.g Novagen. Also, there is a good journal, low impact factor, but specific for this fiels: Journal of Protein Expression & Purification. I check it regularly for new things in the field (generally there are tricks you can learn from how people dealt with their specific protein). Also there is a series of protocols: Current Protocols in Protein Science, or Current Protocols in Molecular Biology. They are written book style but they are mainly online. Actually, this is a field that, in my opinion is based on knowledge, not experience. There is a certain practical experience one needs, but if one lacks the basic knowledge of what is what, he can do the same mistake over and over again.

Andreea

[carlmartin]

Yes, been going from one page to another in openwetware.org. Some things make sense and others are totally foreign. Same goes for the genotype notation am looking at Biobricks at the moment and already scratching my head.

Thank you so much for the explanations, Andreea. I appreciate that.

Have a great day!

[ascacioc]

A note on the two promoters situation (I asked a friend yesterday over lunch about this intersting question of yours and she had some additions):
-in the consideration that you are using a vector with its own promoter to clone a gene with natural promoter, and the gene is from the same organism as the vector is i.e. both promoters are recognized by the RNA polymerase that is present in the host, you get a mixture of mRNAs that have the following structure ...vector promoter - vector rbs - original promoter - original rbs - AUGgene... and ...original promoter - original rbs - AUGgene... (rbs = ribosome binding site).
-the rbs is not a precise sequence, it is 6 consequent nucleotides enriched in As and Gs. Moreover, it is 6-8 bases upstream of the AUG
-in the second construct everything is fine
-in the longer construct (which is most probably the abundant one since expression vector promoters are the strongest found in nature to make your host express mostly your protein) you have a different story: there is a long strand of nucleotides from the 5' end up to the original rbs (the right one for the right AUG) that might be, coincidently, detected as a rbs (the odds are that there are a lot of 6 consecutive A/Gs) and then the ribosome will look for a AUG donwstream the fake rbs and start translation. Again the odds are that there are other AUGs out of frame in this long extra strand of nucleotides. This might lead to the wrong protein being translated.

This is why we do not do this:) We don't like to gamble. If we want the original promoter (which sometimes is the case), we clone the gene with the promoter in such a way to get rid of the original promoter in the vector. If we do not insist on the natural promoter (which anyhow is a weak one, most of them are), we clone only the gene.

Andreea

[lyok]

A little bit hijacking this topic, but if I understand it correctly there are 2 vectors: expression vectors and cloning vectors (or strains).

The cloning vectors are vectors that provide the possibility to clone (duplicate the plasmid?) the genes you want (stable transfer of the plasmid during cell division?). I assume these are high copy vectors and vectors that maintain the plasmid very stable in high copy numbers?
While expression vectors are vectors that provide everything for a stable expression, translation?

But what are the molecular differences between those? I noticed you mentioned the the lack of proteases in expression strains and the lack of endonucleases/recombinases in cloning vectors, but are there other specific differences?
(eg: high copy in cloning vs low copy in expression? Or a very strong promoter in the exrepssion vectors?).

[ascacioc]

There are 2 strains you use for the same vector: cloning strain (K strain e.g. DH5alpha, XL1Blue) is the one with mutant dnases and recombinases and expression strain (B strain e.g BL21, Rosseta etc) is the one with mutant proteases.
In the K strain your ligated plasmid would be nicely transformed since there are minimum dnases and recombinases to affect it. These strains grow slow but have great transformation efficiency. They are also very easy to make competent using the Hanahan method which was actually developed for K strains. These strains are used mainly for plasmid production for mini/midi preping.
In the B strain, your protein will be nicely expressed and protected from the proteases, so it can accumulate up to the harvest time. Minimum preoteases to chop your protein up. Grow faster but have terrible transformation efficiency.
The story behind the original B-strains and K-strains is long. Initially, they were doing this old fashion UV-mutation/selection for the nice bacterial strains that people wanted to have. So they obtained strains to suit them but with lots of mutations. Hence, there are quite a few differences on the genome level between the strains. For more details, I recommend:
http://www.ncbi.nlm....pubmed/19765592 (as I said, Studier writes all the nice articles )
The normal workflow from gene to protein in bacteria: PCR your insert with overhangs for the restriction enzymes you want to use; restrict digest the vector and the insert with these enzymes; ligate vector and insert; transform in K strain and plate; check the ontained colonies for the right insert by colony PCR; miniprep a few of them and send for sequencing; once sequence confirmed, transform the plasmid in B-strain and do the expression of your protein in the B-strain.

Now, this is the classical way (details omitted for simplicity; do not take it as a protocol; there are some steps in between that are essential such as PCR clean up...). If you for example want to transform an epPCR library or any kind of library of variants and select based on expression of your protein, then you cannot wait to obtain the plasmid from K strain, you have to go directly to the B-strain. So, in this case, due to the fact that nowadays we have a bit of more knowledge of how to manipulate bacteria, beyond UV, people have modified the classical BL21(DE3) to get BL21-Gold(DE3) (and similar strains) which has the advantage that it is also a mutant of endonucleas A (as K strains are, but this one is done nicely with modern molecular biology methods not UV ) Now, you will be tempted to say, hack, why should I use the long way and not transform the ligation directly to BL21(DE3)-Gold? Well, you could do that, but I personally do not like strains with too many mutations (endonuclease A is needed in the bacteria) because they are not too healthy and stressed bacteria do not produce tons of protein. But this is just me.

Another question that might arise would be: why do you go through K-strain until the expression strain:
1- easy to transform ligation reaction into; if you use BL21(DE3) for transforming you might end up with no clones, but you might also be lucky. The question is: do you want to play the lotery in the lab?
2 - easy to mini/midiprep your plasmid to have enough for sequencing or other things for which you need ug amounts. Again, nobody says you cannot miniprep from BL21, but you need to be good at. I minipreped successfully from B strains, but I am a mixture of luck and good hands:) I have seen people not being able to miniprep from B-strains. So again: how lucky do you feel to do that? You might end up repeating the entire cloning procedure because you were too lazy to go through the K-strain. (we have a saying in my language: the lazy one, runs more; I thought appropriate to insert some wisdom from the elders here )

This was the story about the strains = living organisms that multiplicate on their own. Not to be confused with plasmids: round pieces of DNA that is not able to multiply on its own. For simplicity, I will refer from now own only to bacterial plasmids, even though the parts are common for many other plasmids.
In a plasmid you generally have:
-origin of replication - needed by the bacteria to recognize the plasmid for replication; these origins are that thing that give the copy number; you can have high copy number plasmids (100 copies per cell) or low copy number plasmids (1-10); this is regulated by the origin of replication. In general, for expressing proteins, you want a low copy number plasmid. But for cloning in bacteria a plasmid to be used for mammalian cell culture imaging or for yeast protein expression, basically to be used in another organism, then you use a high copy number plasmid.
-antibiotic resistance - with its own constitutive promoter and terminator; produces protein to clear the antibiotic; makes the bacterium survive in the certain antibiotic used for selection
-promoter - used for transcription of mRNA from your insert
-terminator -stops mRNA transcription
-rbs - ribosome binding site - ribosome recognition site for translation to happen
-MCS - multiple cloning site with various restriction sites for you to put your insert into
These are the minimum components you have on an expression vector in bacteria. On top, you have some other regulatory components. But this is advanced molecular biology I will let you discover on your own

Andreea

[Julio-Claudian]

Hi carlmartin and Andreea,

I've just finished reading and ruminate over what was posted here. I believe you, Andreea, have given us a very clear explanation on the two-promoter scenario [and more]. If you'd be kind enough to give your insight/critique to what I'm about to post next:

• Whenever "gene" is mentioned in the books, is it tacitly understood by all molecular biologists as actually the "open reading frame, ORF"?

I gather from my reading that the ORF contained in a gene will ultimately result in the protein. Depending on the reading frame, it could well be tens of bases before the start codon is encountered.

Am I correct in saying that since we don't want to "gamble" with the outcome , it also follows that when one wishes to express a protein, it is crucial that the 'thing' downstream of the promoter be an ORF? Or else, we'll have a situation similar to the two-promoter scenario in our hands.

The cloning vectors are vectors that provide the possibility to clone (duplicate the plasmid?) the genes you want (stable transfer of the plasmid during cell division?). I assume these are high copy vectors and vectors that maintain the plasmid very stable in high copy numbers?
While expression vectors are vectors that provide everything for a stable expression, translation?

I could be wrong but I understand both cloning and expression vectors as genetic elements independent of their copy number. While the term expression vector seems to bring to mind a vector used for the overproduction of proteins for purification, a cloning vector can be an 'expression' vector by virtue of it having a promoter (err... right?).

In a rescue / complementation experiment, I believe that plasmids such as the pUC or pGEM with their respective promoters and being in the right environment would be expressed and restore the host.

As for stability and maintenance, it does appear to me in the beginning that high copy number is more superior to low copy one but after some digging found that the latter can be stably maintained too.

I quote Nordström and Austin:

Natural bacterial plasmids are inherited with a very high degree of stability. The loss rate of low-copy-number plasmids can be as low as 10^-7 per cell division (57). This implies the existence of mechanisms that control the distribution of the plasmids at cell division.

In general, for expressing proteins, you want a low copy number plasmid. But for cloning in bacteria a plasmid to be used for mammalian cell culture imaging or for yeast protein expression, basically to be used in another organism, then you use a high copy number plasmid.

I guess certain things are just like that . Brings to mind a discussion I had a while back with someone here regarding copy number and the resulting mRNA and protein but that's for another topic.

Cheerio!

[lyok]

In general, for expressing proteins, you want a low copy number plasmid. But for cloning in bacteria a plasmid to be used for mammalian cell culture imaging or for yeast protein expression, basically to be used in another organism, then you use a high copy number plasmid.

I am having problems understanding this: if you want to express a certain gene , then why use a low copy number plasmid?

(I can understand this for proteins that might be toxic for cells... but in general , isnt it better to have a high copy number and thus more DNA and more protein?)

Altough, perhaps we are discussing something different as Julio-Claudian also noticed at the end of his post.


Also the followng part:

There are 2 strains you use for the same vector: cloning strain (K strain e.g. DH5alpha, XL1Blue) is the one with mutant dnases and recombinases and expression strain (B strain e.g BL21, Rosseta etc) is the one with mutant proteases.
In the K strain your ligated plasmid would be nicely transformed since there are minimum dnases and recombinases to affect it. These strains grow slow but have great transformation efficiency. They are also very easy to make competent using the Hanahan method which was actually developed for K strains. These strains are used mainly for plasmid production for mini/midi preping.
In the B strain, your protein will be nicely expressed and protected from the proteases, so it can accumulate up to the harvest time. Minimum preoteases to chop your protein up. Grow faster but have terrible transformation efficiency.

You do mean that in a cloning vector the "inserted" plasmid DNA is more or less "left alone" and not degraded/inserted via (homologous) recombinase while in an expression vector, this plasmid DNA has more change of being degraded/inserted into the genome , right?

So its save to conclude that in cloning vector the main characterisic is that the plasmid DNA is kept stable and not altered.

And that in an expression vector its the protein processing that is kept stable=> no break down of proteins (or less proteins break down)

[phage434]

Cloning strains, such as DH5a, Top10, etc. have important knockouts in specific genes. The recA gene knockout eliminates much of the accidental recombination of the strain. The endA knockout eliminaes an important endonuclease, which can rapidly trash DNA during and after cell lysis. The knockout of the hsdR genes remove restriction enzymes which can cut transformed DNA before it can be methylated. Typically, few of these genes are knocked out in the expression strains, such as BL21. There, important knockouts include the lon protease, which can trash proteins during cell lysis.

[ascacioc]

Wow, the more specific I try to be, you guys raise more questions Sorry for taling so long to come back to you.

I will start with Julio-Claudian:

I am personally not the most careful person with nomenclature. I state this is the beginning to make you aware that I might be off with some things. I understand by ORF the sequence from the start codon to the stop codon. Some people include in the definition of the ORF the stop codon, others do not include. Hence, ORFs code for proteins.

For the question related to is ORF=gene, I will give you a riddle (actually is the fact that made me also think about this a few years back): C. elegans has ~20000 different proteins but ~36000 genes. Where does the difference come from? You (all of you except phage434) should attempt an answer. You will receive the answer after at least one attempt is done. I do this because if I give you all the answers, it goes in through one ear, and out through the other. Thinking it yourself will make you never forget the answer.

However, most of us refer to genes as those DNAs coding for proteins.

If you figure out the answer for the above riddle, you will realize that not always downstream the promoter you need an ORF.
Hint: promoter/terminator are needed for transcription; rbs, start/stop codons are needed for translation. Think about the product of each process.
Reading your comment, I have to make clear that you need some space between promoter, rbs, ORF, terminator. For some of them you even need specific distances e.g. rbs has to be 6-8 bases upstream the start codon. So, it is crucial to have the optimum distances in order to have your protein expressed. Thankfully, the people who sell those expression vectors have it all figured out for us to clone it directly in the MCS and be sure that our protein will be expressed. Usually we don't bother ourselves with this type of questions: we take care that we have only the ORF from ATG to TAA (no additional promoters ) and we insert this in the MCS. BTW: sometimes when you clone in commercial vectors using the MCS you get some additional amino acids at the termini, which are not the end of the world. It took me a while until I gave up trying to clone my inserts in such a way that I do not have this problem. (this appears especially when you try to clone in vectors that add a tag to your protein). But let's keep on discussing for the sake of understanding what's happening in our experiments (don't think that I understand everything happening on the molecular level, I am still learning myself, I can only help with what I know)
Cloning vector vs expression vector: yes you are right; the difference is in having the promoter, rbs, terminator. I never used a cloning vector. The synthetic gene companies send the genes under the form of gene inserted in a cloning vector. Also some people working in generating DNA libraries are using cloning vectors because it is easier to handle (they are smaller, easier to transform, do not express the proteins from the genes, which make the bacteria happier).
With the origin of replication: I do not know why for expression you use the low copy number plasmids. I have just noticed that all my expression vectors have the low copy number plasmids. The reason that was given to me is that having too many copies per cell would lead to overproduction of the protein which is toxic to the cell. However, having strong promoters such as T7 has the same result. But maybe it is better not to change to many variables at once. For example, when I test which plasmid is better for my protein, I check different promoter strengths (among other things). If I would have different stregths of replication origins, I would change 2 variables in between experiments at once and I wouldn't know what is the cause for the difference.

Edited by ascacioc, 12 August 2012 - 03:35 PM.

[ascacioc]

@ lyok:
You seem to confuse strains and vectors: in the cloning vector, as Julio-Claudian observed, there is no promoter for your gene. Take care, strains are the living things with all the proteins/enzymes, DNA, RNA and a lipid membrane around while the vectors are the circular DNA. I know what you mean, but others might miss the message if we do not call the things what they are
Except naming bacteria vectors, you got it right.

[ascacioc]

@phage434
I have a question regarding what you said:

The endA knockout eliminaes an important endonuclease, which can rapidly trash DNA during and after cell lysis. ... expression strains, such as BL21. There, important knockouts include the lon protease, which can trash proteins during cell lysis.

For both of them you say that the degradation of DNA/protein can happen during the cell lysis. What keeps them from degrading it in the cell? I always thought that the only one that is acting upon cell lysis is the OmpT since it is the outer membrane protease while lon is found in the cytosol. Moreover, I thought that lon protease is degrading the aggregated proteins in the cytosol, among other important functions. This is why, I assumed that if you have too many proteins (like in the case of overexpression), lon might degrade them.

Andreea