Dave Gray's Session 1 Email Q&A

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The following is the text of my email to Lisa Scheifele (I incorrectly addressed her as "Liz") following our first "Build a Gene" session:


This is David Gray from the BUGGS lab class/project. I have been going back over my notes and wonder if you could address some questions when you have time.

Hi David, I tried to answer your questions as best as I could below. They were very insightful and I think/hope that we will discuss and further explain many of the steps that you asked about as we progress through the project.

First, I would like to get a clearer picture of the project. As I understand it, we are planning to insert a segment of DNA that codes for EmGFP into some other cell (was it yeast?). Looking up info about EmGFP, the first one I find is taken from jellyfish. Is that what we will be using?

So as I understand it, once we have (hopefully) successfully spliced the EmGFP DNA into the DNA of the (yeast?) cell, that will be used to create proteins that will fluoresce green - I assume under black light. Is that correct?

Yes, exactly. We will be inserting a variant of the jellyfish GFP gene into bacteria (E. coli), which should make them fluoresce green under UV light. The original GFP gene was isolated from jellyfish and then was modified in the lab to create many different color variants, including the emerald variant that we are creating. Since then, fluorescent proteins have also been isolated from other organisms such as sea corals and they can be expressed in other cell types as well. More good reading here: http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html

On Saturday, we amplified the vectors and promoters. The primers are encoded bits of DNA that tell the enzymes where to begin the duplication process. We also added the DNA templates that the promoters will attach to and replicate with. Is the DNA template a full segment of DNA from, say, a jellyfish? Or is it an extracted segment of DNA from a jellyfish? Or was it specifically constructed DNA that simply includes the code required to generate the EmGFP protein? Since we referred to our templates as a "vector template" and "promoter template", it seems likely that it has been custom constructed in a way that will allow itself to integrate into the target cell's DNA given that is the purpose of a "vector" (http://en.wikipedia.org/wiki/Vector_(molecular_biology)) and that sounds like something different from standard cell function.

The vector is a lab engineered piece of DNA. Of the vector types that the Wikipedia article discusses, we are using a plasmid vector. The vector does not yet carry any jellyfish genes, but once we create our EmGFP gene, we will join its DNA with the vector DNA and the promoter DNA to produce a recombinant DNA molecule. The vector will then carry the inserted EmGFP gene and promoter into the bacterial host cell (a process termed transformation, which we will perform in the 4th class), and the bacterial cell will transcribe and translate the EmGFP gene to make fluorescent EmGFP protein.

I have in my notes that GFP variants are about 750 neucleotides and that we ordered 60 neucleotide sets (20 pieces). As I look at that now, it doesn't make sense. 60 * 20 = 1200. So that doesn't seem to correlate to the 750 number. How do these numbers relate? Why was it that we ordered these in smaller lengths? And how do they get reassembled into the correct 750 neucleotides? (Sorry if I have the picture completely wrong. I thought it made sense at the time…)

Exactly! The combined length of the 20 pieces is always longer than that of the final assembled gene. This is due to how we assemble the pieces together and is an essential property of synthetic gene assembly. We’ll talk about it next time-didn’t want to have too much information the first day

This may be too much to ask, but I am interested in understanding more of how the vector goes about inserting itself into the genome of the target cell - if in fact it does. (The alternative is that it might coexist with in the cell, acting sort of like an extra chromosome and duplicating in parallel with the native DNA.) If it does insert itself, how does it decide where to do that? Perhaps there are some materials that you know of on the web that discuss this at an adequate level of detail to be meaningful without requiring too deep a familiarity with the terminology of genetics to be comprehensible.

The plasmid vectors that we are working with don’t actually insert into the DNA of the host cell, instead they are maintained in the bacterial cell outside of the native chromosome, but replicated along with the chromosome. There are other types of plasmid vectors that do insert into the cell-they do this either by inserting into random locations in the host cell DNA or because they contain a DNA sequence that is identical to a sequence of DNA in the host cell genome which directs where they insert (http://novella.mhhe.com/sites/0070070017/student_view0/biology_1/chapter_20/integration_and_excision_of_a_plasmid.html)

I'm also interested in why the promoter is needed and how it goes about doing its work. I may be able to find that info online, but if you wish to weigh in, I would welcome it.

The promoter is necessary to get the DNA that encodes the gene (in our case EmGFP) transcribed into a messenger RNA. It’s where RNA polymerase, the enzyme that makes the messenger RNA, binds the DNA just before the start of the gene. The promoter is also crucially important because it is often highly regulated by both positive and negative factors (gene “switches”) that can both increase or decrease the level of mRNA that is produced and therefore the amount of protein that is produced. We therefore tend to classify promoters as “strong” (because they cause abundant transcription of their gene) or “weak” because they cause low levels of transcription of the gene. http://www.hhmi.org/biointeractive/gene-switch

During the break in class, we discussed how a primer could be used to extract a specific segment of DNA, working from both ends. The first step in the process is to separate the two halves of the DNA by heating it to near the boiling point of water. My next question what how the primer attaches in this scenario. I assume that as the liquid cools to the point that the DNA can attach to the primer, it could equally well attach to its other half. Perhaps that doesn’t happen because once separated, the two halves are highly unlikely to be properly aligned and in close enough proximity to reattach. Also, depending on the number of primer stands in the mix, it may be that the odds are much greater that the DNA will come in contact with one of them rather than to the other strand. Perhaps after separating, the DNA doesn't have a strong "desire" to get back together given that in normal cell replication, the two halves don't rejoin but are constructed into separate double helixes. Anyway, can you clear this up?

This is just a matter of the relative ratios of the primers and the templates as you suggest. A genome is 106 to 109 nucleotides long while a primer is only 20 nucleotides. Therefore small amounts of primer (in terms of mass) actually contain many many copies. The abundance of primer is always designed to be far in excess of the amount of template DNA in a PCR reaction, thereby maximizing the chance that when two DNA strands anneal, it will favor attachment of primer and template rather than two strands of the template.

As I understand it, "GFP" stands for "Green Fluorescent Protein". But it comes in many colors, unless I'm mistaken. So is "GFP" used for all the colors because green was the first, naturally occurring protein that all the other colors are based on?

GFP should only be used for the original gene and other green variants, such as EGFP (enhanced GFP), EmGFP (emerald GFP). The other colors are usually termed RFP (red fluorescent protein), YFP (yellow fluorescent protein), CFP (cyan fluorescent protein), etc.

Seems it's probably a good thing that we have a two week break. I have much to investigate!

If you would like to put off responding to any of these points until we can discuss them face-to-face at the next BUGGS session, that's fine too. And if there are any you think I'll find satisfactory answers to via Google, Wikipedia and such, feel free to say so.

Some sites that I like to use are http://www.nature.com/scitable, http://www.hhmi.org/biointeractive, and http://www.dnalc.org/resources/animations/ (there’s a nice animation of PCR there).

Thanks again for taking the time to lead the BUGGS class!

- Dave Gray

Hope that gives you a good start! Feel free to email me for any more clarifications, it’s no problem.

To Dave Gray's Build-A-Gene Experience Notes