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Why can't we use plasmids to add genes to ourselves?

Why can't we use plasmids to add genes to ourselves?


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Reading these answers I wonder, why doesn't "gene therapy" use self-contained plasmids instead of trying to splice a length into a chromosome?


First, plasmids do not usually integrate into the chromosomes of human cells. In the laboratory, plasmids can be introduced into the cytosol of cells, and plasmid DNA will then be "read" by the host cell and used to make new proteins, for example. But plasmids usually remain separate from the cell's own DNA, and in that sense they are not effective for gene therapy.

Second, our cells are quite good at detecting and destroying plasmids, as well as other foreign DNA. This is a self-defense mechanism, evolved to get rid of viruses and other nasty parasites that might be trying to hijack the cell. In the lab, where we are working with isolated cells in a culture dish, with no immune system, plasmids are typically tolerated. But in a human being, immune cells that have specialized in detecting intruders are very competent at detecting such manipulations, and they will quickly sound the alarm and wreak havoc… So if injected into the body, plasmids are quickly detected and destroyed.

Research in gene therapy aims to find more efficient ways of "editing" the DNA of human cells, ultimately in a way that can be safely applied in the body. Such a technique does not exist yet. The latest CRISPR-Cas9 gene editing system is a phenomenal tool, and is very efficient at getting into the DNA of human cells; but the CRISPR-Cas9 proteins must themselves be delivered into the cell, for which a virus is typically needed, and this would again cause immune reactions if attempted in a human body.


I believe we could use plasmid insertion to assist with gene therapy. EBV can maintain a plasmid in the cells it infects and the plasmids can effectively replicate. With a greater understanding of the latency and the implications of inserting genes using EBV, it may be possible for gene therapy to occur via genes on a plasmid. references: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562867/ http://jvi.asm.org/content/65/1/483.full.pdf


Chapter 3 Genetic Engineering

Genes contain coded information that leads to the production of proteins. Proteins, in turn, are responsible for creating the traits that characterize individual organisms. Therefore, if a way could be found to transfer genes from one organism to another, creatures could be manufactured with traits that they had never before exhibited. Based on the description of the structure of DNA provided by Watson and Crick, researchers began to search for a way to cut genes from the DNA of one organism and paste them into another. By the 1970s, they had the answer, and the science of genetic engineering was born. It was a giant step forward. Now, a mere thirty years later, it is possible to exchange genes between one plant and another and one animal and another. It is even possible to transpose genes between plants and animals. No organism𠅏rom primitive life-forms, like bacteria, to higher order animals, like human beings—is exempt from this genetic swap meet. Genetic engineering has led to monumental advances in medicine and agriculture, but it has also given rise to a storm of controversy and debate over the limits on humankind's intrusion into the natural order of things.


IRA FLATOW: This is Science Friday. I’m Ira Flatow. One of the most powerful tools biologists use today is not some new DNA sequencer. It’s not a gene printer, either. In fact, it’s not something we humans invented at all.

It’s something we borrowed from bacteria, a sophisticated self-defense they have, a molecular machine that chops up invading viruses to stop an infection. The tool is called CRISPR-cas9, and it allow scientists to edit DNA, to modify or delete genes and add new ones, too. It’s already being used to develop new drugs. And its versatility is not without controversy.

Like, should you use CRISPR to edit the genes in a human embryo? Should you use it to modify mosquitoes so they can’t transmit malaria? Lots of big questions.

But before we get ahead of ourselves, my next guest says this gene editing technology, well, it’s not as easy to use or as much of a guaranteed home run as we have been led to believe. Carmela Haynes is an Assistant Professor in the School of Biology and Health Systems Engineering at Arizona State University in Tempe. Welcome back to Science Friday.

CARMELA HAYNES: Hi, Ira. It’s great to be here. Thank you.

IRA FLATOW: You’re welcome. We’ve been reading sort of a challenge about how easy CRISPR is to use. Rudolf Jaenisch of MIT said quote, “any idiot could do it”. And that’s been sort of put to the test and found out that any idiot could really not do it that easily.

CARMELA HAYNES: Right, yeah, definitely. So our lab spends a lot of time studying a system that is unique to any living thing that is basically made up of more than one cell. And so sort of the catch there is that any idiot could do it assuming that there are no roadblocks or special things about how the DNA is coiled up inside of the cell that would prevent the CRISPR– I guess a sort of scissors-like machinery to get in and actually access the target gene.

IRA FLATOW: So for example, in human embryos, those cells are pretty well coiled up?

CARMELA HAYNES: Indeed, yes. And so the thing about the CRISPR machinery is that, I mean it is fantastic. It’s very good at what it does. But over the millions of years that it’s evolved, it’s operated inside bacteria in a space where we don’t see the same, quite the same type of DNA coiling and packaging that we see in a cell like cell in a human embryo.

So the DNA in embryonic cells, in particular, has a lot of interactions deep inside the nucleus that sort of package and separate certain genes away from the rest of the environment. So this actually poses a bit of a molecular challenge to the CRISPR machinery in terms of accessing the target that you’re going after.

CARMELA HAYNES: Whenever– we talk about CRISPR, a lot on the show because it is such a cutting edge technology and one that is fraught with ethical decisions. And the more I talk to people around, they say, you know it sounds pretty easy.

But what you don’t hear of are a lot of failed attempts at using it. A lot of trial and error, and so on. Would you agree with that?

CARMELA HAYNES: I would definitely agree with that. So when scientists publish what get higher priority are those success stories. So if there’s a particular gene that has a lot of importance in medical research, there is a lot of work that’s put into picking just the right spot on that gene that through trial and error, we find that CRISPR is very good at cutting. So what you’ll see in a lot of the publications are examples of where it works.

But due to the way that publications are structured, and our general culture around reporting on successes and not so much failures, there’s a lot of information out in the body of scientific knowledge that tells us that it doesn’t always work quite as well as we expect it to.

IRA FLATOW: It’s CRISPR itself, the whole connection to bacteria is just, it’s so fascinating, isn’t it? That bacteria have been fighting this fight with viruses, every bacteria has a virus that wants to eat it, and has come up with this mechanism to stave it off. And then here we are sort of adopting it.

CARMELA HAYNES: Oh, yes definitely. That is a fascinating aspect of the use of the system. I think that when scientists started figuring out how the system worked, they immediately started thinking about applications. There are things that are similar in the research community, like things called restriction enzymes that are also derived from bacteria that are very good at cutting up DNA.

But I think that another– speaking of defense mechanisms, the cells of higher organisms, as it were, have a system that doesn’t necessarily rely on cutting invading DNA. What cells like human cells like to do is sort of after the DNA finds its way into the nucleus, what has happened in a lot of cases or at least we have a lot of evidence for this is that special proteins that co-exist with the DNA inside of a cell nucleus will take the DNA and package and wrap it in a very tight structure. So that those invading pieces of DNA can’t replicate and excise themselves and move around and sort of mess up our genetic code. So human cells have their own defense mechanism that I think that, in the early stages of developing CRISPR as a tool, we hadn’t quite started to address that challenge just yet.

IRA FLATOW: But you think it is an addressable challenge?

CARMELA HAYNES: Oh definitely. So a little over, just about two years ago, my grad student and I– so a fantastic grad student, Renee Dare, we’d worked with some other professors to start teaching a course on synthetic biology. So that’s my field. We think of ways to put together borrowed bits and pieces from biology and make very useful tools.

So we set out to develop a course at Cold Spring Harbor Laboratory. And we decided to focus on teaching about CRISPR for aspiring synthetic biologists. So we sat down and thought about how to set up this lesson. And we wanted to make it very interesting.

So we started looking in the CRISPR, and we got very curious– since our specialty is in DNA packaging and how to engineer that– we started asking ourselves, well, you know since CRISPR comes from a bacterial system. It’s evolved in an environment where it hasn’t really been exposed to all of these complex packaging machineries. What if we actually challenged CRISPR with a packaged system?

So we happened to have a very nice engineered, human cultured cell line that we use in the lab to probe questions about engineering DNA packaging. And what we did was that we did a pretty simple experiment where we packaged, artificially packaged a target sequence inside the cells. And then introduced CRISPR and measured how good it was at cutting.

And we found that in some cases, you can completely eliminate or completely block CRISPR cutting altogether, if that target gene is packaged. One of the neat things about our artificial system is that it is built around the same machinery that stem cells use to shut down activity of certain genes that are involved in converting the stem cell into a specialized cell like a muscle cell or a nerve cell. So our artificial system, although it is artificial, it overlaps. It uses a lot of the same protein bits and pieces that stem cells use to make their genes inaccessible. Yeah, we think that our finding, which we just published in ACS Synthetic Biology is extremely relevant to sort of looking deeper, more deeply into the challenges facing practical use of CRISPR as a tool.

IRA FLATOW: Let me see if I can get a quick phone call in before we have to go. Let’s go to Panama City, Florida. Matthew, welcome to Science Friday.

MATTHEW: Gosh, thank you, Ira. So how many idiots are using this around the world? To get my mind around it, like there are hundreds of laboratories, are there thousands of laboratories?

IRA FLATOW: Hey, is that a fair– thanks, thanks for the call. Is that a fair question to ask, Carmela?

CARMELA HAYNES: Well, I think the right way to think about that is thinking about the steps involved in using CRISPR yourself. So it is– I do agree that the technology itself is very accessible, like tomorrow if I decided to change my lesson plan here at ASU, I could come up with, I could develop a tutorial that would allow students to look at a publicly available human DNA sequence online at a website like NCBI, and look at a sequence. Teach them a couple of basic rules about how to target CRISPR to a site and give them instructions on how to design a CRISPR.

So this is a customizable system where you can change nucleotide sequences that are associated with the CRISPR machinery to match the target that you wanted to go after and edit. So I could certainly teach those students how it works on paper. We could even walk through, OK, well, how would you order the DNA from a company, a DNA synthesis company to build your own customized CRISPR tool?

But then the huge barrier– you can map everything out on paper. You can run algorithms to look for just the right target, but then when it comes to actually taking cells or an organism and then expressing it and getting what you want, there are a lot of complicated steps involved. So while CRISPR may be accessible to everyone, and it’s easy to learn how to use it, actually implementing it is quite a challenge.

IRA FLATOW: So we’re not going to see a home CRISPR kit for your living room just quite yet. But you never know how soon. Yeah, exactly.

IRA FLATOW: Dr. Haynes, thank you for taking time to be with us today. Carmela Haynes is an Assistant Professor in the School of Biological and Health Systems Engineering at Arizona State University in Tempe, Arizona.


Transformation Of Escherichia Coli With pGLO Plasmid

ABSTRACT:
This experiment focuses on genetic engineering and transformation of bacteria. The characteristics of bacteria are altered from an external source to allow them to express a new trait, in this case antibiotic resistance. In is experiment foreign DNA is inserted into Escherichia coli in order to alter its phenotype. The goal of the experiment is to transform E. coli with pGLO plasmid, which carries a gene for ampicillin resistance, and determine the transformation efficiency.

The bacteria are transformed by a combination of calcium chloride and heat shock. When the bacteria are incubated on ice, the fluid cell membrane is slowed and then the heat shock increases permeability of the membrane. The results obtained in the experiment show that the E. coli that was transformed with pGLO was able to resist ampicillin and grow in its presence. These results suggest that microorganisms can be genetically engineered to selectively resist certain contaminants, which means that they can potentially be used to human benefit to rid the environment, or even the human body, of unwanted toxins.

Transformation occurs when altered genetic characteristics of bacteria are acquired from a different source. Plasmids are used to transform bacteria because they are small pieces of DNA capable of independently replicating and therefore transferring their (often beneficial) traits to the bacteria. The goal of genetic transformation in this experiment is for the bacteria Escherichia coli to obtain an antibiotic resistance to ampicillin, which can be physically observed when the bacteria expresses the reporter gene Green Fluorescent Protein (GFP) because the transformed bacteria will glow green under UV light when in the presence of arabinose. The gene for GFP is naturally found in a bioluminescent jellyfish, allowing it to glow in the dark. The plasmid used to transform the bacteria contains the antibiotic resistance along with a gene that codes for the fluorescent protein.

The objective of this experiment is to genetically engineer ampicillin resistant E. coli by transforming it with a plasmid containing an antibiotic resistance gene and a gene that codes for GFP and to calculate the transformation efficiency. This will alter the phenotype by inserting foreign DNA. This experiment is important because it demonstrates a technique that has practical applications in areas such as environmental concern. Bacteria that have been genetically altered can obtain traits that allow it to be used to break down toxic compounds (i.e. oil spill) to reduce the amount of harmful substances found in soil and water (Spilios, 2013). Some environmental contaminants are actually toxic for microorganisms, like bacteria, and deactivate the cells abolishing their ability to break down the contaminants (Pieper, 2000). For this reason, bacteria that are resistant to specific toxins are preferable and it would be desirable to replicate their DNA– the resistance gene specifically –and use it to create such resistance in other organisms. This research builds directly from the knowledge used in this experiment transforming E. coli.

The hypotheses for this experiment are that the bacteria will be transformed by the plasmid to develop an antibiotic resistance and will grow despite the presence of ampicillin. Additionally, when arabinose sugar is added to the strain of bacteria transformed by the plasmid, it will express GFP and glow green under UV light.

The methods and procedures for transforming bacteria in this experiment were performed according to Spilios (2013). Bacteria strains of E. coli were genetically transformed with a plasmid to carry ampicillin resistance. 250 microliters of a CaCl2 transformation solution were pipetted into closed test tubes, which were then placed on ice. A starter plate with E. coli colonies was observed and two colonies were transferred into the tubes of CaCl2, one colony in each, and dispersed evenly throughout the solution by mixing. The tubes were then placed back on ice. Ten nanograms of pGLO DNA solution was then added to one of the tubes and the tubes were incubated on
ice for ten minutes. While the tubes were incubating, four plates of LB nutrient agar were obtained, one containing just LB, two also with ampicillin, and one also with ampicillin and arabinose. The test tubes after the ten minutes were incubated for 50 seconds in a 42 degree Celsius water bath and then returned to the ice for another two minutes. The tubes were then removed from the ice and 250 microliters of LB nutrient broth was added to each tube, which was then incubated for an additional ten minutes at room temperature. 100 microliters of the solution in the tube containing pGLO was added to one plate with just LB agar and ampicillin and to the plate with ampicillin and arabinose. 100 microliters of the solution in the tube without pGLO was added to the other plate containing LB and ampicillin as well as to the plate containing only LB. The solution was spread around the surface of each agar using a new sterile loop for each plate. The plates were then incubated at 37 degrees Celsius and results were observed and recorded after 27 hours.

RESULTS:
Plate
Observations
Growth
“+pGLO LB/amp”
Yellow colonies still yellow under UV light
Yes
“+pGLO LB/amp/ara”
Yellow colonies glow green under UV light
Yes
Table 1. Transformed Plates After Incubation shows that there was growth of transformed bacteria and their observable characteristics.

Plate
Observations
Growth
“-pGLO LB/amp”
Nothing
No
“-pGLO LB”
Yellow colonies still yellow under UV light
Yes
Table 2. Control Plates After Incubation shows whether there was growth of E. coli without the presence of the pGLO plasmid after exposure to ampicillin.

The transformed bacteria showed growth despite the presence of ampicillin (Table 1), whereas the control plate with ampicillin did not show any growth, and the control plate with only LB agar showed the formation of a lawn of bacteria (Table 2). The transformed bacteria on the plate with LB, ampicillin and arabinose differed from the transformed plate without arabinose in that they glowed green under UV light. The bacteria without arabinose maintained an unaltered appearance under UV light. The transformation efficiency for the transformed bacteria was 5.2 × 104 transformants per microgram of DNA.

DISCUSSION:
In this experiment the objective was to transform E. coli with the pGLO plasmid and calculate the transformation efficiency. The hypotheses were that the plate with only LB agar and untransformed E. coli would grow a lawn the control plate of untransformed bacteria with LB and ampicillin would experience no growth the transformed plate with just LB and ampicillin would grow colonies of bacteria but it would not glow green under UV light and the transformed plate with LB, ampicillin and arabinose would grow colonies that would glow green under UV light. The results found supported each of these hypotheses as the bacteria grew as predicted. The untransformed bacteria grew on the LB plate rapidly as none of them were inhibited by the ampicillin, but they did not grow when exposed to ampicillin on the other control plate because it did not have the gene for antibiotic resistance. The transformed plates both carried the gene to resist ampicillin from the pGLO plasmid, so they were able to grow colonies that were the offspring of the genetically transformed E. coli. The plate that also contained arabinose grew colonies of bacteria that contained the gene that not only resists ampicillin but also expresses GFP and causes the bacteria to glow green under UV light. The arabinose operon in E. coli is a
set of genes that will only become active when the sugar arabinose is present (Spilios, 2013). Genes are transcribed and translated to break down arabinose for energy. The expression of enzymes that break down arabinose can be turned on or off. In the pGLO plasmid, the GFP gene has replaced genes that code for the break down of arabinose. In this experiment, the arabinose added acted as the “on switch” to cause the operon to produce GFP. The transformation efficiency calculated for the results was 5.2 × 104 transformed cells per microgram of DNA used. This is slightly shy of the standard transformation efficiency in standard published research. Hanahan’s (1983) experiment states that he improved on the levels of efficiency observed at standard conditions by 100- to 1000-fold with reported efficiency of 5 × 108 transformants per microgram of plasmid. The type growth or nutrient media does not make a large impact on the transformation efficiency, but the concentrations on components that increase growth rate in the media tend to aid in transformation (Hanahan, 1983). Therefore, it may prove beneficial to incorporate a growth medium with higher concentrations of Mg2+ into the experimental design to achieve slightly higher transformation efficiency. Future experiments may focus on working to increase the transformation efficiency of E. coli (Chung, 1989). This can help decrease costs and increase reliability to replace existing methods. As previously stated, developing a working knowledge and understanding of genetic engineering aids in addressing environmental and health concerns. Microorganisms could be used to target specific contaminants or toxins in the environment or in the human body.

LITERATURE CITED:
Chung CT. 1989. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences of the United States of America, 86:2172-2175.

Hanahan D. 1983. Studies on Transformation of Escherichia coli with Plasmids. Journal of Molecular Biology, 166:557-580.

Pieper DH. 2000. Engineering bacteria for bioremediation. Current Opinion in
Biotechnology, 11.3:262-270.

Spilios K (ed). 2013. Principles of Biology, II. Hayden-McNeil Publishing, Plymouth, MI. Module #6, pp 119-127.


Why can't we use plasmids to add genes to ourselves? - Biology

Feeling blue? These bacteria are too &ndash and that makes us molecular cloners feel blue because it means our gene didn&rsquot get into the circular piece of DNA called a plasmid which we put in there, so the bacteria can&rsquot make copies of the gene for us. But the good news is, the blue-white screening system makes it easy to tell &ndash so we can avoid the blue and instead choose a white colony, where molecular cloning went well!

An overview in rhyme, then it&rsquos details time! If the plasmid vector your insert hacks, blue-color-making protein the bacteria lacks! The plasmid has the sequence the bacteria needs, but you wouldn&rsquot know this if your cloning succeeds. When you insert the gene, that sequence you break & the functional protein the bacteria never make. If the colony&rsquos white you know all went right, but if the colony&rsquos blue, you need to redo

Each of the dots on this plate is a clump of genetically-identical bacteria, which we call a colony. They&rsquore genetically identical because bacteria reproduce by doubling their insides (including their DNA) and then splitting in half, giving a full set of everything to each daughter cell.

In addition to their own DNA, these bacteria have an &ldquoextra&rdquo circular piece of DNA called a plasmid. I know that these bacteria have the plasmid I want because the plasmid has antibiotic resistance genes that allow it to survive even though I spiked the bacterial food (media) with the corresponding antibiotics. This is a form of SELECTION &ndash only bacteria with the plasmid inside can grow on these plates.

But, apart from allowing for selection against random bacteria trying to grow on our plates, the plasmid is really just there to act as a vehicle, or VECTOR, to allow us to insert &ldquoany&rdquo gene we want into the bacteria &ndash so, for example, they can make lots of copies of it for us. Since we&rsquore recombining pieces of DNA when we do the vector/insert combo a form of RECOMBINANT DNA. And since the bacteria make lots of copies of it, and when they divide each daughter cell is a genetic clone of the parent cell, we call this general process MOLECULAR CLONING.

The basic gist of molecular cloning is: stick a gene you want to study into a circular piece of DNA called a plasmid vector & stick that plasmid into bacteria to make more of that gene &/or the protein it codes for (possible because the genetic code is universal so any organism can read it). It&rsquos a powerful tool that&rsquos revolutionized molecular biology & biochemistry. BUT it doesn&rsquot always work right, so we need to check that

  1. the bacteria actually took in the plasmid AND
  2. our gene actually got inserted into the plasmid

We can use SELECTION MARKERS like antibiotic resistance genes to check for the presence of the plasmid (1) e.g. if plasmid has Ampicilin (Amp) resistance gene but host bacteria don&rsquot, only bacteria that have the plasmid can grow in presence of Amp,sSo any colonies of bacteria that grow have the plasmid. BUT does that plasmid have your gene in it (2)?You still don&rsquot know, but there are a few ways to find out.

A couple options we&rsquove looked at are the DIAGNOSTIC DIGEST (aka analytical digest) http://bit.ly/30Npa8o & COLONY PCR http://bit.ly/39cADmK . These either CUT (in the digest) or COPY pieces of (in the PCR) the plasmid differently if your gene is present or absent and this gives you different size DNA pieces. In order to see these pieces you have to separate them by size using agarose gel electrophoresis. And for the digest, you have to purify the plasmid DNA before you can even test it. So, while these methods work (for the most part), they can take a while.

BLUE-WHITE SCREENING (B-W screening) is a way to check for presence of an insert without even having to touch the colonies! You just have to look!

It uses protein called BETA-GALACTOSIDASE (B-gal), which is an enzyme that catalyzes (speeds-up) the hydrolysis (water-based breaking up) of LACTOSE (a disaccharide (2 linked sugar units)) into the monosaccharides (individual sugar units) GLUCOSE & GALACTOSE. The GENE for B-gal is called lacZ.

B-gal functions as a homotetramer (4 identical copies stuck together). First it forms dimers (pairs) & then those pairs pair to form tetramers. You need all four because parts of the different copies &ldquocross over&rdquo to contribute to the &ldquoactive sites&rdquo where the functioning occurs

This pair-pairing (dimer to tetramer) occurs using the beginning part of the protein (N-terminus). if you remove this part, dimers can still form, BUT tetramers can&rsquot, so functional protein can&rsquot be made. BUT you can restore this beginning, by adding the needed part, the alpha (a) peptide, back &ldquoin TRANS&rdquo (as a separate molecule). This is known as a-complementation.

In B-W SCREENING you use bacterial host cells have a shortened B-gal (LacZ&Deltam15) that&rsquos missing some of the protein&rsquos first 41 amino acid &ldquoletters&rdquo (11&mdash41). (&Delta is the Greek letter &ldquodelta&rdquo and, in protein/gene jargon, it&rsquos often used to indicate missing parts). These LacZ&Deltam15 are missing the N-terminus part & only have the &ldquoleftover&rdquo &omega-fragment that can&rsquot form tetramers.

To make it form tetramers, it needs those first letters &ndash the &ldquo&alpha-peptide&rdquo &ndash & the plasmid has that to offer in trans (plasmid&rsquos way of paying rent?). So you need both in order to make B-gal. But how do you know if B-gal is actually being made?

B-gal&rsquos usual products (glucose & galactose) aren&rsquot colored, so we can&rsquot see them. BUT B-gal mostly cares about the galactose part & isn&rsquot too picky about what&rsquos on the other side of the glycosidic bond. So we can swap the glucose for something that will give us a product we can see.

X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) has galactose connected to 5-bromo-4-chloro-3-hydroxyindole. &beta-gal splits it up into galactose & 5-bromo-4-chloro-3-hydroxyindole which dimerizes (pairs up) & oxidizes to 5,5&prime-dibromo-4,4&prime-dichloro-indigo which is insoluble & BLUE! So the colony turns blue.

That doesn&rsquot seem too useful though&hellip We only want to grow colonies with our plasmid in the first place, which is why our plasmid ALSO has a selection marker in the form of an antibiotic resistance gene. So just having plasmid-containing colonies turn blue would be redundant &ndash it&rsquod tell us what we already know &ndash the plasmid&rsquos there.

The real power of blue-white screening is making them NOT BLUE. To do this you use plasmids designed to have a multiple cloning site (MCS) (the place you put in your gene) INSIDE the gene for the &alpha-peptide. This way, inserting your gene interrupts the &alpha-peptides&rsquos gene, so the bacteria can&rsquot make functional &alpha-peptide -> can no longer complement the &omega-fragment -> can&rsquot make functional B-gal -> can&rsquot make blue product from X-gal -> colony remains white

  • WHITE COLONIES have insert (in right place)
  • BLUE COLONIES don&rsquot have insert or have it in the wrong place

⚠️whiteness indicates that SOMETHING got inserted. BUT you don&rsquot know that that something&rsquos the something you want -you&rsquod have to sequence it to be sure

Of course, this B-W screening only works if &beta-gal is made! & bacteria don&rsquot want to waste time & resources making &beta-gal if they can&rsquot use it &ndash so we have to trick them into using it

When lactose isn&rsquot around, a REPRESSOR protein sits on & &ldquohides&rdquo the part of the &beta-gall gene (lacZ) where the DNA-to-RNA copier RNA POLYMERASE (RNA Pol) binds to start making an mRNA copy of the gene that can then get read by ribosomes and translated into the protein. So, NO lactose -> no &beta-gal mRNA made -> no &beta-gal made (not even the monomers&hellip)

BUT when lactose is present, B-gal &ldquomoonlights&rdquo as a transglycosylator &ndash it converts lactose into ALLOLACTOSE by changing the glycosidic linkage site between the glucose & galactose units (just shifts things around a little to give you an isomer of lactose &ndash same atoms, different linking). Allolactose binds the repressor causing the repressor to change shape (undergo a conformational change) & fall off -> RNA Pol binds -> &beta-gal mRNA made -> &beta-gal made

So you&rsquod think, why not just add some lactose, or allolactose if we want to do B-W screening? Problem is, &beta-gal can also hydrolyze (split) our de-repressor allolactose. So, we&rsquod have to constantly add more if we wanted to have continuous &beta-gal making. So, instead, we trick the bacteria by using IPTG Isopropyl (&beta-D-1-thiogalactopyranoside). This is an allolactose analog (mimic) that can also de-repress the lac promoter, so &beta-gal is made. BUT unlike lactose it doesn&rsquot get hydrolyzed b y&beta-gal, so it sticks around.

note: if you move the lac promoter in front of a gene for something else, you can use IPTG to trick the bacteria into making that something else on cue. we looked at such &ldquoinduction&rdquo in yesterday&rsquos post on recombinant protein expression in bacteria: http://bit.ly/3cPVhLO

So, in practice, it works something like this.

  1. put your gene in the plasmid. (molecular cloning step)
  2. put the plasmid in the bacteria (transformation step)
  3. plate the bacteria on an agar plate (you&rsquore classic &ldquoPetri dish&rdquo w/food, antibiotic, IPTG, & X-gal
  4. stick it in a nice warm incubator
  5. wait for the bacteria to grow
  6. wait for blue color to show

If you come back the next day & all your colonies are white, don&rsquot get too excited yet &ndash It takes a while (16-20h) for &beta-gal to be expressed (remember it has to get de-repressed & everything first) & get to work. So the blue starts showing up gradually. Sometimes it&rsquos hard to tell early on, but the more you work w/this system, the more of a &ldquosense&rdquo you get for it. The white ones usually look kinda &ldquodifferent&rdquo &ndash they grow bigger & look &ldquogoopier&rdquo

If you come back the next next day & all the colonies are still white, definitely don&rsquot get excited &ndash get suspicious, because cloning is rarely that efficient. Did you forget the IPTG? the X-gal?

note: X-gal is the &ldquoclassic&rdquo chromogenic (color-producing) B-gal substrate, but you can change up that indoxyl part to change the absorbance and therefore the color you see. Our lab uses Bluo-gal, which is really similar but doesn&rsquot have the chlorine . This gives a darker blue product that&rsquos easier to see. You can also do things like move the Cl to a different position to get Magenta-gal (5-bromo-6-chloro-3-indolyl-&beta-D-galactopyranoside). Remove the Br from that and you get the more salmon-colored (thus aptly named) Salmon-gal (S-gal, 6-chloro-3-indolyl-&beta-D-galactopyranoside). The color differences come from the different molecular arrangements absorbing different wavelengths of light. And if you want to know more: http://bit.ly/2RDLVY4

Technical terminology talk time: Color chemistry-ly speaking, &ldquoDYE&rdquo is usually used for *soluble* colored things whereas &ldquoPIGMENT&rdquo implies *nonsoluble*. Since X-gal&rsquos product is insoluble, it&rsquod technically be a *pigment* not a *dye* but in biology, we tend to use the terms interchangeably

Technical terminology talk time 2: B-W is a form of SCREENING as opposed to SELECTION. SELECTION (like w/antibiotic resistance gene) does all the work for you (all the products are &ldquogood&rdquo in the aspect you selected for) BUT w/a screen you have to do some work (even if it&rsquos just looking) &ndash the &ldquoduds&rdquo are still there, they&rsquore just easier to see.

It&rsquos like you&rsquore looking to hire some people &ndash with screening, you go through all the apps & separate them into yes/no piles but keep both for someone else to deal with. With selection, you toss the no pile, so the next person only sees the yeses.

Now for a couple caveats: Firstly, screening isn&rsquot always yes/no. You can have &ldquomaybes&rdquo like if you&rsquore screening for drugs that might work for something & some work really well, some don&rsquot work at all, but some are &ldquopotentials&rdquo

Secondly, only certain bacterial hosts & plasmids are designed for blue-white screening. Some that are:


Geographic Isolation Drives Evolution Of Hot Springs Microbe

Sulfolobus islandicus, a microbe that can live in boiling acid, is offering up its secrets to researchers hardy enough to capture it from the volcanic hot springs where it thrives. In a new study, researchers report that populations of S. islandicus are more diverse than previously thought, and that their diversity is driven largely by geographic isolation.

The findings open a new window on microbial evolution, demonstrating for the first time that geography can trump other factors that influence the makeup of genes an organism hosts.

S. islandicus belongs to the archaea, a group of single-celled organisms that live in a variety of habitats including some of the most forbidding environments on the planet. Once lumped together with bacteria, archaea are now classified as a separate domain of life.

"Archaea are really different from bacteria &ndash as different from bacteria as we are," said University of Illinois microbiology professor Rachel Whitaker, who led the study.

Whitaker has spent almost a decade studying the genetic characteristics of S. islandicus. The new study, in the Proceedings of the National Academy of Sciences, compares three populations of S. islandicus, from hot springs in Yellowstone National Park, Lassen National Park in California and the Mutnovsky Volcano on the Kamchatka Peninsula, in eastern Russia.

The extreme physical needs of S. islandicus make it an ideal organism for studying the impact of geographic isolation. It can live only at temperatures that approach the boiling point of water and in an environment that has the pH of battery acid. It breathes oxygen, eats volcanic gases and expels sulfuric acid. It is unlikely that it can survive even a short distance from the hot springs where it is found.

By comparing the genetic characteristics of individuals from each of the three locations, Whitaker and her colleagues were able to see how each of the S. islandicus populations had evolved since they were isolated from one another more than 900,000 years ago.

The complete genetic package, or genome, of S. islandicus contains a set of core genes that are shared among all members of this group, with some minor differences in the sequence of nucleotides that spell out individual genes. But it also contains a variable genome, with groups of genes that differ &ndash sometimes dramatically &ndash from one subset, or strain, to another.

Whitaker's team found that the variable genome in individual strains of S. islandicus is evolving at a rapid rate, with high levels of variation even between two or three individuals in the same location.

"Some people think that these variable genes are the way that microbes are adapting to new environments," Whitaker said. "You land in a new place, you need a new function in that new place, you pick up that set of genes from whoever's there or we don't know who from, and now you can survive there. We have shown that does not occur."

"This tells you that there's a lot more diversity than we thought," Whitaker said. "Each hot spring region has its own genome and its own genome components and is evolving in its own unique way. And if each place is evolving in its own unique way, then each one is different and there's this amazing diversity. I mean, beetles are nothing compared to the diversity of microbes."

Archaea, like bacteria, can transfer genes to one another, but they also obtain new genes from free-floating genetic elements, called plasmids, or from viruses that infect the cells and insert their own genes into the archaeal DNA. What did vary in the genomes of S. islandicus could be traced back to plasmids and viruses, Whitaker said. There were also a lot of lost genes, with much variation in the genes lost between the strains.

"Most of the genes that are coming and going, at least on Sulfolobus, seem to be on viruses and plasmids," Whitaker said. The researchers found that about one-third of the variable genes were specific to a geographic location. The viruses and plasmids that had lent their genes to Sulfolobus in one site were different from those found in another. Also, much of the variation was found in genes devoted to the microbe's immune system, indicating that S. islandicus is evolving largely in response to the assault of local pathogens such as viruses.

These findings challenge the idea that microbes draw whatever they may need from a near-universal pool of available genetic material, Whitaker said. It appears instead that S. islandicus, at least, acquires new genes from a very limited genetic reservoir stored in viruses and other genetic elements that are constrained to each geographic location on Earth.


To Sing Fung and Ding Xiang Liu
Vol. 73, 2019

Abstract

Human coronavirus (HCoV) infection causes respiratory diseases with mild to severe outcomes. In the last 15 years, we have witnessed the emergence of two zoonotic, highly pathogenic HCoVs: severe acute respiratory syndrome coronavirus (SARS-CoV) and . Read More

Figure 1: Taxonomy of HCoVs: the updated classification scheme of HCoV and other coronaviruses. The six known HCoVs are in blue. Abbreviations: BtCoV, bat coronavirus BuCoV, bulbul coronavirus HCoV.

Figure 2: Genome structure of human coronaviruses (HCoVs). Schematic diagram showing the genome structure of six known HCoVs (not to scale). The 5′-cap structure (5′-C) and 3′-polyadenylation (AnAOH-3.

Figure 3: Replication cycle of human coronaviruses (HCoVs). Schematic diagram showing the general replication cycle of HCoVs. Infection starts with the attachment of HCoVs to the cognate cellular rece.

Figure 4: Induction and modulation of autophagy by HCoV infection. Schematic diagram showing the signaling pathway of autophagy and the modulatory mechanisms utilized by HCoV. Viruses and viral compon.

Figure 5: Apoptosis induced by HCoV infection and modulatory mechanisms. Schematic diagram showing the signaling pathway of intrinsic and extrinsic apoptosis induction and the modulatory mechanisms ut.

Figure 6: Induction and modulation of unfolded protein response by HCoV infection. Schematic diagram showing the three branches of UPR signaling pathway activated and regulated by HCoV infection. Viru.

Figure 7: Activation and modulation of MAPK signaling pathways by HCoV infection. Schematic diagram showing the activation and modulation of MAPK signaling pathway by HCoV infection. Viruses and viral.

Figure 8: Type I interferon induction and signaling during HCoV infection and modulatory mechanisms. Schematic diagram showing the induction and signaling pathways of type I interferon during HCoV inf.


Cloning DNA into a vector, step by step

To introduce foreign DNA into a circular vector, scientists carry out a three-step process:

Scientists first remove their gene of interest from the DNA sequences on either side of it. They can use restriction enzymes to do the cutting. These enzymes, which came originally from bacteria, cut DNA at specific sites in the sequence. If there’s not enough DNA for successful cutting or no suitable restriction enzyme recognition sites around the gene, scientists first use polymerase chain reaction (PCR) to make many more copies. By designing their PCR primers carefully, they can introduce new restriction sites on either side of the copied DNA sequence.

Opening up the vector

Next, scientists make a cut in the circular DNA sequence of the vector. They use the same restriction enzymes as they used to cut out the gene in step 1. This turns the vector into a linear molecule and makes it ready to accept the new piece of DNA.

Sticking the vector and the gene together

The final step in cloning is to incorporate the DNA of interest into the vector. Scientists mix the gene and the opened vector together with a bacterial enzyme called DNA ligase . The ligase sticks DNA ends together to form a single circular molecule that includes both the vector and the gene.

Find out more in these articles:


How the DNA Revolution Is Changing Us

The ability to quickly alter the code of life has given us unprecedented power over the natural world. Should we use it?

If you took a glance around Anthony James’s office, it wouldn’t be hard to guess what he does for a living. The walls are covered with drawings of mosquitoes. Mosquito books line the shelves.

Hanging next to his desk is a banner with renderings of one particular species—Aedes aegypti—in every stage of development, from egg to pupa to fully grown, enlarged to sizes that would even make fans of Jurassic Park blanch. His license plates have a single word on them: AEDES.

“I have been obsessed with mosquitoes for 30 years,” says James, a molecular geneticist at the University of California, Irvine.

There are approximately 3,500 species of mosquito, but James pays attention to just a few, each of which ranks among the deadliest creatures on Earth. They include Anopheles gambiae, which transmits the malaria parasite that kills hundreds of thousands of people each year. For much of his career, however, James has focused on Aedes. Historians believe the mosquito arrived in the New World on slave ships from Africa in the 17th century, bringing with it yellow fever, which has killed millions of people. Today the mosquito also carries dengue fever, which infects as many as 400 million people a year, as well as such increasingly threatening pathogens as chikungunya, West Nile virus, and Zika.

In a widening outbreak that began last year in Brazil, Zika appears to have caused a variety of neurological disorders, including a rare defect called microcephaly, where babies are born with abnormally small heads and underdeveloped brains.

The goal of James’s lab, and of his career, has been to find a way to manipulate mosquito genes so that the insects can no longer spread such diseases. Until recently, it has been a long, lonely, and largely theoretical road. But by combining a revolutionary new technology called CRISPR-Cas9 with a natural system known as a gene drive, theory is rapidly becoming reality.

CRISPR places an entirely new kind of power into human hands. For the first time, scientists can quickly and precisely alter, delete, and rearrange the DNA of nearly any living organism, including us. In the past three years, the technology has transformed biology. Working with animal models, researchers in laboratories around the world have already used CRISPR to correct major genetic flaws, including the mutations responsible for muscular dystrophy, cystic fibrosis, and one form of hepatitis. Recently several teams have deployed CRISPR in an attempt to eliminate HIV from the DNA of human cells. The results have been only partially successful, but many scientists remain convinced that the technology may contribute to a cure for AIDS.

In experiments, scientists have also used CRISPR to rid pigs of the viruses that prevent their organs from being transplanted into humans. Ecologists are exploring ways for the technology to help protect endangered species. Moreover, plant biologists, working with a wide variety of crops, have embarked on efforts to delete genes that attract pests. That way, by relying on biology rather than on chemicals, CRISPR could help reduce our dependence on toxic pesticides.

No scientific discovery of the past century holds more promise—or raises more troubling ethical questions. Most provocatively, if CRISPR were used to edit a human embryo’s germ line—cells that contain genetic material that can be inherited by the next generation—either to correct a genetic flaw or to enhance a desired trait, the change would then pass to that person’s children, and their children, in perpetuity. The full implications of changes that profound are difficult, if not impossible, to foresee.

“This is a remarkable technology, with many great uses. But if you are going to do anything as fateful as rewriting the germ line, you’d better be able to tell me there is a strong reason to do it,” said Eric Lander, who is director of the Broad Institute of Harvard and MIT and who served as leader of the Human Genome Project. “And you’d better be able to say that society made a choice to do this—that unless there’s broad agreement, it is not going to happen.”

No discovery of the past century holds more promise—or raises more troubling ethical questions.

“Scientists do not have standing to answer these questions,” Lander told me. “And I am not sure who does.”

CRISPR-Cas9 has two components. The first is an enzyme—Cas9—that functions as a cellular scalpel to cut DNA. (In nature, bacteria use it to sever and disarm the genetic code of invading viruses.) The other consists of an RNA guide that leads the scalpel to the precise nucleotides—the chemical letters of DNA—it has been sent to cut. (Researchers rarely include the term “Cas9” in conversation, or the inelegant terminology that CRISPR stands for: “clustered regularly interspaced short palindromic repeats.”)

The guide’s accuracy is uncanny scientists can dispatch a synthetic replacement part to any location in a genome made of billions of nucleotides. When it reaches its destination, the Cas9 enzyme snips out the unwanted DNA sequence. To patch the break, the cell inserts the chain of nucleotides that has been delivered in the CRISPR package.

By the time the Zika outbreak in Puerto Rico comes to an end, the U.S. Centers for Disease Control and Prevention estimates that, based on patterns of other mosquito-borne illnesses, at least a quarter of the 3.5 million people in Puerto Rico may contract Zika. That means thousands of pregnant women are likely to become infected.

Currently the only truly effective response to Zika would involve bathing the island in insecticide. James and others say that editing mosquitoes with CRISPR—and using a gene drive to make those changes permanent—offers a far better approach.

Gene drives have the power to override the traditional rules of inheritance. Ordinarily the progeny of any sexually reproductive animal receives one copy of a gene from each parent. Some genes, however, are “selfish”: Evolution has bestowed on them a better than 50 percent chance of being inherited. Theoretically, scientists could combine CRISPR with a gene drive to alter the genetic code of a species by attaching a desired DNA sequence onto such a favored gene before releasing the animals to mate naturally. Together the tools could force almost any genetic trait through a population.

Last year, in a study published in the Proceedings of the National Academy of Sciences, James used CRISPR to engineer a version of Anopheles mosquitoes that makes them incapable of spreading the malaria parasite. “We added a small package of genes that allows the mosquitoes to function as they always have,” he explained. “Except for one slight change.” That change prevents the deadly parasite from being transmitted by the mosquitoes.

“I’d been laboring in obscurity for decades. Not anymore, though—the phone hasn’t stopped ringing for weeks,” James said, nodding at a sheaf of messages on his desk.

Combating the Ae. aegypti mosquito, which carries so many different pathogens, would require a slightly different approach. “What you would need to do,” he told me, “is engineer a gene drive that makes the insects sterile. It doesn’t make sense to build a mosquito resistant to Zika if it could still transmit dengue and other diseases.”

To fight off dengue, James and his colleagues have designed CRISPR packages that could simply delete a natural gene from the wild parent and replace it with a version that would confer sterility in the offspring. If enough of those mosquitoes were released to mate, in a few generations (which typically last just two or three weeks each) entire species would carry the engineered version.

James is acutely aware that releasing a mutation designed to spread quickly through a wild population could have unanticipated consequences that might not be easy to reverse. “There are certainly risks associated with releasing insects that you have edited in a lab,” he said. “But I believe the dangers of not doing it are far greater.”

It has been more than 40 years since scientists discovered how to cut nucleotides from the genes of one organism and paste them into the genes of another to introduce desired traits. Molecular biologists were thrilled by the possibilities this practice, referred to as recombinant DNA, opened for their research. From the start, however, scientists also realized that if they could transfer DNA between species, they might inadvertently shift viruses and other pathogens too. That could cause unanticipated diseases, for which there would be no natural protection, treatment, or cure.

This possibility frightened no one more than the scientists themselves. In 1975, molecular biologists from around the world gathered at the Asilomar Conference Grounds, along California’s central coast, to discuss the challenges presented by this new technology. The group emerged from the meeting having agreed to a series of safeguards, including levels of laboratory security that escalated along with the potential risks posed by the experiments.

It soon became clear that the protections seemed to work and that the possible benefits were enormous. Genetic engineering began to improve the lives of millions. Diabetics, for example, could count on steady supplies of genetically engineered insulin, made in the lab by placing human insulin genes into bacteria and then growing it in giant vats. Genetically engineered crops, yielding more and resisting herbicides and insects, began to transform much of the world’s agricultural landscape.

Yet while genetically engineered medicine has been widely accepted, crops produced in a similar fashion have not, despite scores of studies showing that such products are no more dangerous to eat than any other food. As the furor over the labeling of GMOs (genetically modified organisms) demonstrates, it doesn’t matter whether a product is safe if people refuse to eat it.

CRISPR may provide a way out of this scientific and cultural quagmire. From the beginning of the recombinant era, the definitions of the word “transgenic” and the term “GMO” have been based on the practice of combining in a laboratory the DNA of species that could never mate in nature. But scientists hope that using CRISPR to alter DNA could appease the opposition. It gives researchers the ability to redesign specific genes without having to introduce DNA from another species.

Golden rice, for example, is a GMO engineered to contain genes necessary to produce vitamin A in the edible part of the grain—something that doesn’t happen naturally in rice plants. Each year up to half a million children in the developing world go blind for lack of vitamin A—but anti-GMO activists have interfered with research and prevented any commercial production of the rice. With CRISPR, scientists could almost certainly achieve the same result simply by altering genes that are already active in rice plants.

Scientists in Japan have used CRISPR to extend the life of tomatoes by turning off genes that control ripening. By deleting all three copies of one wheat gene, Caixia Gao and her team at the Chinese Academy of Sciences in Beijing have created a strain that is resistant to powdery mildew.

Without regulation, the tremendous potential of this revolution could be overshadowed by fear.

Farmers have been adjusting genes in single species—by crossbreeding them—for thousands of years. CRISPR simply offers a more precise way to do the same thing. In some countries, including Germany, Sweden, and Argentina, regulators have made a distinction between GMOs and editing with tools such as CRISPR. There have been signs that the U.S. Food and Drug Administration might follow suit, which could make CRISPR-created products more readily available and easily regulated than any other form of genetically modified food or drug. Whether the public will take advantage of them remains to be seen.

The potential for CRISPR research to improve human medicine would be hard to overstate. The technology has already transformed cancer research by making it easier to engineer tumor cells in the laboratory, then test various drugs to see which can stop them from growing. Soon doctors may be able to use CRISPR to treat some diseases directly.

Stem cells taken from people with hemophilia, for example, could be edited outside of the body to correct the genetic flaw that causes the disease, and then the normal cells could be inserted to repopulate a patient’s bloodstream.

In the next two years we may see an even more dramatic medical advance. There are 120,000 Americans on waiting lists to receive organ transplants, and there will never be enough for all of them. Thousands of people die every year before reaching the top of the list. Hundreds of thousands never even meet the criteria to be placed on the list.

For years, scientists have searched for a way to use animal organs to ease the donor shortage. Pigs have long been considered the mammal of choice, in part because their organs are similar in size to ours. But a pig’s genome is riddled with viruses called PERVs (porcine endogenous retroviruses), which are similar to the virus that causes AIDS and have been shown to be capable of infecting human cells. No regulatory agency would permit transplants with infected organs. And until recently, nobody has been able to rid the pig of its retroviruses.

Now, by using CRISPR to edit the genome in pig organs, researchers seem well on their way to solving that problem. A group led by George Church, a professor at Harvard Medical School and MIT, used the tool to remove all 62 occurrences of PERV genes from a pig’s kidney cell. It was the first time that so many cellular changes had been orchestrated into a genome at once.

When the scientists mixed those edited cells with human cells in a laboratory, none of the human cells became infected. The team also modified, in another set of pig cells, 20 genes that are known to cause reactions in the human immune system. That too would be a critical part of making this kind of transplant work.

Church has now cloned those cells and begun growing them in pig embryos. He expects to start primate trials within a year or two. If the organs function properly and are not rejected by the animals’ immune systems, the next step would be human trials. Church told me he thinks this could happen in as few as 18 months, adding that for many people the alternative to the risk of the trial would surely be death.

Church has always wanted to find a way to provide transplants for people who aren’t considered healthy enough to receive them. “The closest thing we have to death panels in this country are the decisions made about who gets transplants,” he said. “A lot of these decisions are being made based on what else is wrong with you. Many people are rejected because they have infectious diseases or problems with substance abuse—a whole host of reasons. And the conceit is that these people would not benefit from a transplant. But of course they would benefit. And if you had an abundance of organs, you could do it for everyone.”

The black-footed ferret is one of the most endangered mammals in North America. Twice in the past 50 years, wildlife ecologists assumed that the animals, which were once plentiful throughout the Great Plains, had gone extinct. They came close every black-footed ferret alive today descends from one of seven ancestors discovered in 1981 on a cattle ranch near Meeteetse, Wyoming.

But the ferrets, inbred for generations, lack genetic diversity, which makes it harder for any species to survive.

“The ferrets are a classic example of an entire species that could be saved by genomic technology,” said Ryan Phelan of the group Revive & Restore, which is coordinating efforts to apply genomics to conservation. Working with Oliver Ryder at the San Diego Frozen Zoo, Phelan and her colleagues are attempting to increase the diversity of the ferrets by introducing more variable DNA into their genomes from two specimens preserved 30 years ago.

Phelan’s work can address two immediate and interlocking threats. The first is lack of food: Prairie dogs, the ferrets’ main prey, have been decimated by sylvatic plague, which is caused by the same bacterium that gives rise to bubonic plague in humans. And the plague is also fatal to the ferrets themselves, which become infected by eating prairie dogs that have died of the disease. A vaccine against human plague developed in the 1990s appears to provide lifelong immunity in ferrets. Teams from the Fish and Wildlife Service have captured, vaccinated, and released as many of the ferrets (a few hundred exist in the wild) as they can. But such a ferret-by-ferret approach cannot protect the species.

A more sophisticated solution has been proposed by Kevin Esvelt, an assistant professor at the MIT Media Lab, who developed some of the CRISPR and gene drive technology with Church. Esvelt describes his work as sculpting evolution. “All you need to do is provide resistance,” he explained—by encoding antibodies generated by vaccination and then editing them into the ferrets’ DNA.

With gene drives and CRISPR
we now have a power over species of all kinds that we never thought possible.

Esvelt believes a similar approach could not only help the ferrets resist plague but could also help eradicate Lyme disease, which is caused by a bacterium transmitted by ticks that commonly feed on white-footed mice.

If resistance to Lyme could be edited into the mice’s DNA with CRISPR and spread through the wild population, the disease might be reduced or eliminated with little visible ecological impact. Esvelt and Church, however, both feel strongly that no such experiment should be attempted without public participation and unless the scientists who carry it out have developed a reversal system, a kind of antidote. Should the original edits have unforeseen ecological consequences, they could drive the antidote through a population to cancel them out.

Black-footed ferrets are hardly the only endangered animals that could be saved through a CRISPR gene drive. The avian population of Hawaii is rapidly disappearing, largely because of a type of malaria that infects birds. Before whalers brought mosquitoes in the early 19th century, birds in the Hawaiian Islands had no exposure to the diseases that mosquitoes carry, and therefore no immunity. Now only 42 of more than a hundred species of birds endemic to Hawaii remain, and three-quarters of those are listed as endangered. The American Bird Conservancy has referred to Hawaii as “the bird extinction capital of the world.” Avian malaria is not the only threat to what remains of Hawaii’s native birds, but if it cannot be stopped—and gene editing seems to be the best way to do that—they will likely all disappear.

Jack Newman is a former chief science officer at Amyris, which pioneered development of a synthetic form of artemisinin, the only genuinely effective drug available to treat malaria in humans. Now he focuses much of his attention on eliminating mosquito-borne disease in birds. The only current method of protecting birds from malaria is to kill the mosquitoes by spreading powerful chemicals over an enormous region. Even that is only partially successful.

“In order to kill a mosquito,” Newman says, “the insecticide actually has to touch it.” Many of these insects live and breed deep in the hollows of trees or in the recessed crags of rock faces. To reach them with insecticides almost certainly would require poisoning much of the natural life in Hawaii’s rain forests. But gene editing, which would result in sterile mosquitoes, could help save the birds without destroying their surroundings. “Using genetics to save these species is just an incredibly targeted way to address a variety of environmental ills,” Newman says. “Avian malaria is destroying the wildlife of Hawaii, and there is a way to stop it. Are we really willing to just sit there and watch?”

In February of this year, U.S. Director of National Intelligence James Clapper warned in his annual report to the Senate that technologies like CRISPR ought to be regarded as possible weapons of mass destruction. Many scientists considered the comments unfounded, or at least a bit extreme. There are easier ways for terrorists to attack people than to conjure up new crop plagues or deadly viruses.

Nevertheless, it would be shortsighted to pretend that the possibility for harm (including, and perhaps especially, accidental harm) does not exist with these new molecular tools. The scientists most responsible for advances like CRISPR agree that when we begin to tinker with the genetic heritage of other species, not to mention our own, it may not be easy, or even possible, to turn back.

“What are the unintended consequences of genome editing?” asked Jennifer Doudna, as we spoke in her office at the University of California, Berkeley, where she is professor of chemistry and molecular biology. In 2012, Doudna and her French colleague Emmanuelle Charpentier were the first to demonstrate that scientists could use CRISPR to edit purified DNA in lab dishes. “I don’t know that we know enough about the human genome, or maybe any other genome, to fully answer that question. But people will use the technology whether we know enough about it or not.”

The more rapidly science propels humanity forward, the more frightening it seems. This has always been true. Do-it-yourself biology is already a reality soon it will almost certainly be possible to experiment with a CRISPR kit in the same way that previous generations of garage-based tinkerers played with ham radios or rudimentary computers. It makes sense to be apprehensive about the prospect of amateurs using tools that can alter the fundamental genetics of plants and animals.

But the benefits of these tools are also real, and so are the risks of ignoring them. Mosquitoes cause immense agony throughout the world every year, and eradicating malaria or another disease they carry would rank among medicine’s greatest achievements. Although it is clearly too soon to contemplate using CRISPR in viable human embryos, there are other ways of editing the human germ line that could cure diseases without changing the genetic lineage of our species.

Children born with Tay-Sachs disease, for instance, lack a critical enzyme necessary for the body to metabolize a fatty waste substance found in the brain. The disease is very rare and occurs only when both parents transmit their defective version of the gene to a child. With CRISPR it would be easy to treat one parent’s contribution—say, the father’s sperm—to ensure that the child did not receive two copies of the faulty gene. Such an intervention would clearly save lives and reduce the chance of recurrence of the disease. A similar outcome can be achieved already through in vitro fertilization: Implanting an embryo free of the defective gene ensures that the child won’t pass the disorder on to a future generation.

When faced with risks that are hard to evaluate, we have a strong tendency to choose inaction. But with millions of lives at stake, inaction presents its own kind of danger. Last December scientists from around the world met in Washington to discuss the difficult ethics of these choices. More discussions are planned. There will never be simple answers, but without any regulatory guidance—and there is none yet for editing human DNA—the tremendous potential of this revolution could be overshadowed by fear.

“With gene drives and CRISPR we now have a power over species of all kinds that we never thought possible,” says Hank Greely, director of Stanford’s Center for Law and the Biosciences. “The potential good we can do is immense. But we need to acknowledge that we are dealing with a fundamentally new kind of power, and figure out a way to make sure we use it wisely. We are not currently equipped to do that, and we have no time to lose.”


Why can't we use plasmids to add genes to ourselves? - Biology

When comparing prokaryotic cells to eukaryotic cells, prokaryotes are much simpler than eukaryotes in many of their features (Figure 1). Most prokaryotes contain a single, circular chromosome that is found in an area of the cytoplasm called the nucleoid.

Practice Question

Figure 1. A eukaryote contains a well-defined nucleus, whereas in prokaryotes, the chromosome lies in the cytoplasm in an area called the nucleoid.

In eukaryotic cells, DNA and RNA synthesis occur in a separate compartment from protein synthesis. In prokaryotic cells, both processes occur together. What advantages might there be to separating the processes?

The size of the genome in one of the most well-studied prokaryotes, E.coli, is 4.6 million base pairs (approximately 1.1 mm, if cut and stretched out). So how does this fit inside a small bacterial cell? The DNA is twisted by what is known as supercoiling. Supercoiling means that DNA is either under-wound (less than one turn of the helix per 10 base pairs) or over-wound (more than 1 turn per 10 base pairs) from its normal relaxed state. Some proteins are known to be involved in the supercoiling other proteins and enzymes such as DNA gyrase help in maintaining the supercoiled structure.

Eukaryotes, whose chromosomes each consist of a linear DNA molecule, employ a different type of packing strategy to fit their DNA inside the nucleus (Figure 2). At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The histones are evolutionarily conserved proteins that are rich in basic amino acids and form an octamer. The DNA (which is negatively charged because of the phosphate groups) is wrapped tightly around the histone core. This nucleosome is linked to the next one with the help of a linker DNA. This is also known as the “beads on a string” structure. This is further compacted into a 30 nm fiber, which is the diameter of the structure. At the metaphase stage, the chromosomes are at their most compact, are approximately 700 nm in width, and are found in association with scaffold proteins.

In interphase, eukaryotic chromosomes have two distinct regions that can be distinguished by staining. The tightly packaged region is known as heterochromatin, and the less dense region is known as euchromatin. Heterochromatin usually contains genes that are not expressed, and is found in the regions of the centromere and telomeres. The euchromatin usually contains genes that are transcribed, with DNA packaged around nucleosomes but not further compacted.

Figure 2. These figures illustrate the compaction of the eukaryotic chromosome.