Science Buddies: "Ask an Expert"

Hi,

I am inserting a second copy of a gene (cpdA) into E. coli K-12 strain, which codes for a specific phosphodiesterase. I am doing to this to increase the concentrations of the phosphodiesterase in the cell, which will set off a chain of reactions within the cell. First I am going to extract DNA from the E. coli cells, then I will amplify the gene using the PCR process, and then I am going to insert the gene into a plasmid called the pGLO plasmid. This plasmid is developed for a high school level classroom lab made available by bio-rad, and it contains an ampicillin- resistant gene and many restriction sites for different restriction enzymes.
Here is a link to the map of the plasmid:

http://www.bio-rad.com/B2B/BioRad/produ ... ources#map


The link looks killer, but it should work.

Inserting the gene into the plasmid is the step that is giving me so much trouble. I am pretty sure what I want to do is cut the plasmid with restriction enzymes so that instead of being a circular strand it is long segment with sticky ends on each end. Then I want to cut the amplified cpdA gene’s ends with some sort of exonuclease that will make the ends of the cpdA gene compliment the ends of the plasmid that is now a segment. Assuming those steps are correct, I have run into a problem. The sticky ends of the cpdA gene won’t compliment any of the sticky ends of the plasmid I can create with different endonucleases (Eco RI, HindIII, etc.). So I thought to fix this, maybe I can design my primers so that in the PCR process the gene will be amplified along with the nucleotides on either side of it. That way I can add on as many nucleotides as I want until I find restriction sites that will match up with a possible sticky end on the plasmid strand. (by add on, I mean amplify addition necleotides on the sides of the cpdA gene that are naturally next to the gene in the E coli genome)

Do think that any of my thinking is incorrect at all, or especially if I cannot just “add on� nucleotides to the section of DNA being amplified in PCR? Can you think of a more direct or easier way to insert the gene into the plasmid?

Please tell me if any of my explanation does not make sense by the way I said it.

Your method of adding bases to the cpdA amplification sounds logical and feasible. One thing to consider, however: Do you know if the DNA to either side of the cpdA gene codes for other enzymes? If it is just "junk" DNA there shouldn't be a problem adding bases in the amplification, but you may want to be careful if it is "coding" DNA. You won't want another gene than your target ones expressed.

Sonia

how can I tell if the bases next to the gene are coding for something or just junk DNA? Do I have to go through and figure out the amino acid sequence?

Here is a link I found that shows the genes around cpdA:

http://biocyc.org/ECOLI/NEW-IMAGE?type= ... ject=G7579

If you put your cursor over each gene, it will tell you what the gene codes for and how far it is from the surrounding genes. The in-between distances are the "junk" DNA.

Hope this helps!

Sonia

So because there is no junk DNA between cpdA gene and the yqiA gene, you don't think I can "add on" the nucleotides to the beginning of the cpdA gene that will be amplified. Is this correct? I thought that I could still do that as long as there isn't a start codon in the added on DNA from the yqiA gene that will cause it to start transcribing the codes for that protein. Even so, shouldn't there be a stop codon at the very end of the yqiA gene that will prevent it form screwing up the protein synthesis using the cpdA gene?

You're right that protein synthesis can't begin without a START codon. Since the START codon for yqiA is a good distance away from cpdA, it should be fine to add base pairs to that side. However, you will want to be careful to limit the base pairs that you add to the other side of cpdA so you don't include the START codon for yqiB.

Hi mmb11,

Looks like this project is rocking!

I think your plan to incorporate restriction enzyme sites into the primers for amplifying cpdA is a good one. Bear in mind that the restriction enzymes will not cut right at the end of your PCR product so you will need to add 4-8 nucleotides before the restriction enzyme sequence. You can find out more about how close to the end the enzyme will cut at NEB
http://www.neb.com/nebecomm/tech_refere ... efault.asp

its also a good idea to pick enzymes that will cut at the same temmperature and in the same buffer if you can. otherwise you need to think about which to cut with first.

I also think you have made a good choice with your vector. It looks like you can cut out the GFP gene in it and replace it with cpdA and use the promoter that is in the vector to drive production of cpdA. The promoter is "inducible' so the cpdA will not be switched on until you add arabinose to the medium. So you could try some different conditions with different amounts of arabinose.

When you have figure out which restriction site you are going to use you can post your new primers and we can check them if you like.

good job,

Caroline

Ok. I know this is a basic thing to be able to do, but I just can't get genome sequences off of Pub Med. I am looking for the E coli k-12 and cpdA gene sequence. can someone help me out?

Hi mmb,

Pubmed isn't the friendliest website but I guess its hard to make things easy to find when they have so much data.

To find the full genome sequence for E.coli K12 starting from the Pubmed homepage (http://www.pubmed.gov which actually redirects to another site) first select "Genome" from the pulldown menu. (Its starts off showing "pubmed" and genome is about the 8th choice on the pulldown) then type Escherichia coli K12 into the box and hit Go.
Then scroll down to the 11th hit on the list of results (not sure why it isn't number one but at least its there) . This goes to the summary of the genome. Click on the RefSeq link to go to the full genome sequence.
It will take a while to load.
WHile you have it in Genbank format you can do a search of the page (using control F or tool under edit from your browser) for the gene name to find the section of the anotation that talks about your gene of interest. And gives the coordinates of where the gene is located and which strand it is on (complement or the sequence given)
I would recommend getting the sequence in FASTA format. You can do this by selecting with the pulldown menu on the second row (4th option).

I think Linh had mentioned another webiste at the University of Wisconsin in one of our early posts. That would be another option as well rather than the convoluted way I just described!

-Caroline

Hi there,

You could try the Profiling of the E. coli Chromosome (PEC) website - see link below. I typed in cpdA in the search area and got a hit - the gene on this site is called icc, but cpdA is one of the alternative names so I think it is the same thing. Once you find the gene just click on it and lots of info including both DNA and amino acid sequence comes up.

http://www.grs.nig.ac.jp/ecoli/pec/index.jsp

Hope this helps.

Mary

Caroline:

I am sorry. It has been a long time since I have taken a look at my poject. It seems I really have the whole deal with restriction enzymes incorporated into my primers all screwed up. You said:

Bear in mind that the restriction enzymes will not cut right at the end of your PCR product so you will need to add 4-8 nucleotides before the restriction enzyme sequence.

OK, since the Primers are obviously going to have to correspond with the sequence of the cpdA gene and the n'tides around it, I don't see how I can just "add on" a restiction site 4-8 bases from the gene. I was just figuring on looking at the n'tide sequence around the gene in the entire E coli K-12 genome and finding the nearest restriction site for the specific endonuclease that I want to use (say Eco R1 or Hind III or something).

I interpreted your quote as saying you can just incorporate a restriction site onto the end of your primer without reguard for the actual n'tides next to the gene in the E coli genome. So say the restiriction site was GGCAT and the gene that is being amplified is ATTGACT, you would end up with something like "GGCATATTGACT" with a few extra n'tides in there like you said. But where I got stuck was you can't just add on restriction sites because the n'tides next to the gene may not be "GGCAT". That may not make too much sense the way I explained it.

Thank you,

Maggie

Hi Maggie,
Good to hear from you again.

I think maybe my explanation was a bit confusing.
What I was aiming for was to suggest that you make a forward primer with this format:
NNNN-Restriction site-ATGstart of your gene of interest

then reverse primer will have
complimentary end of your gene-different restriction site-NNNN

does that make more sense?

-Caroline

But how will the primers with that format amplify my gene if the restriction sites in my primer don't correspond with the n'tides next to my gene? Or are you implying that I find restriction enzymes with R sites that correspond with the n'tides next to my gene?

I have a feeling that I missing a big chunk of info on restriction sites and enzymes, because I just don't see how this is going to work.

Hi Maggie,

The 3' end of the primer is the most important part. If you think of a zipper, there is often that little bit at the top that doesn't knit together but the zip still stays up. Its kind of like that. The 3' part of the primer (about 18 bases) is an exact match and will bind on your gene of interest and the end part with the restriction site and the few extras will hang of the end, like the top of the zipper. The polymerase will bind where the primer and your gene of interest are annealed together and will amplify your gene. The same thing will happen on the other strand with your other primer as long as the 3' end is complimentary.

Then you will get products that have your gene surrounded by the restrictions sites and the few extra bases and you can cut that product with the enzymes.
You also cut the vector. Purify away the enzymes and then "ligate" or join the products using the sticky ends of the restriction sites.

-Caroline

Caroline:

I thank you ao much! You answered my question perfectly. Thank you so much!!!

I have redesigned my primers so that it has two extra nucleotides, then an Eco RI restriction site, and finally 11 n'tides from the gene I am amplifying.

My forward primer is GC;GAATTC;TTGGAAAGCCT

My reverse primer is AAGGCTACTGA;GAATTC;GG

They both have an annealing temperature of 56*C. I have already made sure that the 5'->3' versions of these primers do not occur in any place in the E coli genome.

Have I covered all my bases?

Hi Maggie,

Your pun about "have i covered all of my bases" made me smile.

I havent had a chance to check these thoroughly yet but I would recommend using different restriction enzymes in each primer if you have more than one site in the vector. Why - because if you have the same restriction sites at each end of your PCR product the product can ligate into the vector in either direction. Approximately half will have the correct orientation with your gene going forward but half will have it backwards and then it wouldnt make the protein.

I would probably add another couple of nucleotides at each end just to be sure there is enough room to cut.

One very imported thing i had forgotten to remind you to check yet is that EcoRi (and the other ennzyme you choose) does not cut inside your gene of interest. There is a tool at the NEB site that you can use for this
http://tools.neb.com/NEBcutter2/index.php

Sorry how it seems like there is always something else. But its better to try and think of eveything beforehand rather than getting the wrong size products and then having to figure out why.

-Caroline

Caroline:

I looked through the map of the plasmid I am using and found 7 different restiction enzyme sites that only occur once in that plasmid and do not occur inside of my gene.
You said:
[quotei]ts also a good idea to pick enzymes that will cut at the same temmperature and in the same buffer if you can. [/quote]

Unfortunately all of these enzymes cut at a temp of 37*C, with the exception of one which cuts at 25*C. This is not even close to the annealing temperation I want of 55-65*C. What can I do about this?

Hi Maggie,

The restriction digest is done in a separate reaction, after you have purified the PCR product so doesn't need to be a compatible temperature with the PCR.

So I would pick 2 that cut at 37C. I would try and pick restriction sites that are downstream of the pBAD promoter and cut in the GFP part of pGLO. What are the 7 you can choose from?

-Caroline

I was thinking about it, and it would defeat the purpose of my project to insert my gene in a way that would make it an arabinose operon. For my results, I need a gene that is always being transcribed, not just when arabinose is available.

Maggie

Hi Magige,

I don't think there is another promoter in the pGLO that you can use so you would have to find another vector if you wanted to have a constitutive promoter (one that is always active).
Or you could just keep the inducible one switched on all of the time by making sure that there is excess arabinose in the medium.

-Caroline

OK, now I feel very sheepish. Is a promoter really nessecary to transcribing the gene and beginning protein synthesis? I thought the polymerase just attatched to the gene at the start codon to start the transcription. Yeah, I feel like a fool.

Hey Maggie,

Don't get down on yourself. This is a really advanced project - I have known assistant professors that had difficulty getting their gene of interest cloned how they wanted it!

Sometimes it hard for me to know what you have already learned about and what I need to tell you. Thats the difficulty with doing this online. You shouldnt feel bad about not knowing something that no one has taught you.

There are different things needed for transcription and protein synthesis. With protein synthesis it starts at the start codon but for transcription there are additional things needed - the promoter. I am a little fuzzy about exactly what is needed in bacteria as I work with eukaryotic cells, but
Wikipedia seems to have a decent explanation about what is needed for transcription in prokaryotes/bacteria.
http://en.wikipedia.org/wiki/Promoter

best of luck,
caroline