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A Hello from Robert Englund to my Molecular and Microbial Techniques Class

Robert Englund on getting 'ready to be Freddy' Krueger | CBC Archives

“Most of my nightmares involve me forgetting my lines in a stage play.”

 
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Posted by on September 23, 2020 in Uncategorized

 

Technicalities of cloning

We live in a time when cloning a gene is a trivial matter done by technicians in the lab using tools that have been around for decades. However, this does not mean that it can be accomplished without knowing precisely how the biology works.

It is often said, even in molecular biology texts, that the DNA of the target gene and a plasmid vector simply need to be cut with the same restriction enzyme so that their ‘sticky ends’ can come together and be ligated into a new construct. This is often diagrammed as a cut and paste procedure (below), suggesting that no further attention to detail is required.

There are two issues (the bulleted items) I have with such a simplified cartoon:

  • This cartoon does not indicate any preference for the direction that the insert takes once it is incorporated into the plasmid.

Inserts have directionality. A gene has a start codon on one end and a stop codon on the other. These must be in line with the promoter sequence and polyA signals that are typically not a part of the insert, but are contributed by the plasmid.

The Promoter sequence is required for binding of the DNA-Dependent RNA-Polymerase that will make the mRNA. This must be upstream of the start codon, ATG. The Poly Signal is responsible for attaching untemplated ‘A’ residues to the 3′ end of the mRNA. It must be downstream of the stop codon, [TAA/TAG/TGA].

The easiest way to prevent this problem is to ensure directional cloning by cutting each end of the vector and insert with different enzymes that don’t possess compatible ends.

The second easiest way to control for cloning your insert in the wrong way is to have a digest with another enzyme to screen for the proper orientation after miniprepping multiple clones. In the figure below, we can see that there is one XhoI site within the insert and one within the plasmid. Because the one in the insert is offset to one side, the size of the fragments generated by this digestion will depend on the orientation of the insert. Knowing which fragment size is associated with proper orientation of the insert will help us to quickly screen our minipreps by XhoI digestion.

  • This cartoon suggests that the recombinant plasmid is the only product of this ligation reaction.

The other problem with this cartoon is that it assumes that the desired ligation of one insert with one plasmid is the most abundant product, when in fact, it probably occurs in a tiny minority of cases. More likely, the plasmid curls up on itself and religates without the insert being incorporated.

The reason that the plasmid religates is because the two ends of the plasmid can religate and they’re close enough together that they probably run into one another more often than an insert does. The way to prevent this is to recall the chemistry involved in this ligation reaction.

This reaction requires a 3′ hydroxyl group and a 5′ phosphate group to come together to form the phosphodiester bond. This happens on each strand of the DNA and is mediated by a ligase enzyme.

However, only one strand being ligated is sufficient to hold the recombinant plasmid together until it can be fixed inside the cell by DNA repair mechanisms. If you remove the phosphate from the plasmid molecule, it cannot come together and be ligated to itself. Instead, only when an insert is incorporated are there the phosphates required to ligate one strand at either end.

The phosphates can be removed from the plasmid DNA using the enzyme, Calf Intestinal Phosphatase (CIP). The only thing you need to remember is that the CIP must be destroyed at the end of its reaction with the plasmid. Otherwise, it can go on to also dephosphorylate the insert DNA when that is added for a ligation reaction. Luckily, CIP is easily inactivated by heat (NEB suggests 80 C for 2 minutes).

An alternative to CIP is Shrimp Alkaline Phosphatase (SIP), also available from NEB.

Using either of these two techniques individually will result in an abundance of your preferred recombinant product.

Happy cloning!

 
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Posted by on September 21, 2020 in Uncategorized

 

What antibody testing for SARS-CoV-2 may get us

The ultimate goal of the new and emerging SARS-CoV-2 antibody tests is to determine when and how we can safely get back to work and restart our stalled economies. While knowing how many people are sick is valuable in tracking the rate of disease spread and identifying people who need to isolate or seek treatment, it misses out on telling us who has had the virus and has recovered (or may have not been symptomatic at all). The idea is that people with antibodies may be protected from (re-)infection and are therefore safe to return to “normal” life. (caveats are that these antibodies are actually protective and will last, conferring immunity).

lateral flow

A good article discussing what antibodies are and how these tests operate was recently published in USA Today. The form of many of these tests is very similar to home pregnancy tests (lateral flow ELISAs), enabling quick, rapid results with no special training to perform.

Eventually, once enough people have been exposed to the virus and recovered or been vaccinated against the virus (probably early next year), then we will have herd immunity sufficient to protect the vulnerable people around us from being exposed to a life-threatening illness.

In the meantime, we’ll probably have to be content with a ‘new normal’ of living in a way that doesn’t prevent the spread of infection but limits it to a level that our hospitals can bear.

 
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Posted by on April 21, 2020 in Uncategorized

 

Cytokine Release and its Attenuation

This is an excellent graphic from the laboratory of Carl June, who is a pioneer in CAR T Cell therapy at UPenn (Moore and June. Science  17 Apr 2020). One of the problems associated with CAR T Cell therapy is the resulting cytokine story often accompanying such a large infusion of immune cells. A similar problem is seen in COVID-19 patients (as well as in those infected with SARS-CoV and MERS-CoV), where it is thought to be the actual cause of death in infected patients.

Preliminary trials using drugs interfering with IL-6 signaling are showing signs of hope for severely sick COVID-19 patients.

Thank you to Michael McHeyzer-Williams (@mmw_lmw) of the Scripps Institute for his tweet pointing me toward this paper.

 

Screen Shot 2020-04-19 at 12.20.59 PM

 
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Posted by on April 19, 2020 in Uncategorized

 

Tired of COVID-19

I have to admit, I’m getting a little bit tired of COVID-19. Or more specifically, I am getting tired of all the social distancing and not being able to work from my campus office. More than anything, this comes out in my frustration at having to use online meetings to conduct classes and student one-on-ones. Perhaps I’m not as effective a communicator as I would like to be, but I miss being able to sketch out ideas on paper to show students how systems work or using a whiteboard to supplement PowerPoint slides.

I am very much looking forward to exposure testing (an antibody test – most likely laminar flow, e.g. pregnancy tests) for SARS-cov-2 to demonstrate that we’ve either had the virus or are otherwise immune to it so we can get back to work!

contagion11

I suggest that the Whitehouse coronavirus taskforce spend some time watching ‘Contagion’ for a roadmap of how we can return to a normally functioning society after an outbreak.

Another front in the fight against this virus that I am eagerly awaiting is a vaccine such as that made by the company Moderna, which uses RNA-like molecules to deliver protein-encoded messages to our cells. I’m eager to see the results of this vaccine’s trials (now at Emory University) both for the immediate effect in preventing COVID-19 as well as to see how this new vaccine platform performs.

I have my fingers crossed that I can get back to work as usual and, equally important, get back to my climbing gym, RoKC.

 

 

 

 
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Posted by on April 2, 2020 in Uncategorized

 

Complement in a Nutshell

Complement is an ancient component of our innate immune system that was initially discovered in the 19th century and named for its ability to complement antibody in the lysis of cells.

In the simplest of terms, complement is triggered by one of three mechanisms (Antibody Triggers the Classical Pathway; Carbohydrates Trigger the Lectin Pathway; The Alternative Pathway is triggered spontaneously.) Once triggered, a cascade of events leads to the assembly of a C3 Convertase, which breaks C3 into the soluble anaphylatoxin C3a and the insoluble C3b, which precipitates onto the surface of the cell and forms a component of additional C3 convertase, thus amplifying the reaction, and also a C5 convertase.Screen Shot 2020-02-17 at 1.53.41 PM

C5 is then digested into an additional, more powerful soluble anaphylatoxin, C5a, and the insoluble C5b, which cooperates with other components to lead to the formation of the Membrane Attack Complex (MAC) comprised of C9 molecules inserted into the plasma membrane.

The anaphylatoxins, C3a, C5a (and, to a lesser extent, C4a), function to induce contraction of the smooth muscle and then an increase in vascular permeability of the capillaries. They further increase the expression of adhesion molecules on these same vascular epithelial cells so as to recruit immune cells to the location. Finally, they promote receptor-mediated chemoattraction of leukocytes.

In summary, complement can be activated through three somewhat distinct pathways, each one converging at a C3 Convertase. Complement will lead to direct cell death via pore formation (MAC complex formation), it will recruit leukocytes to the area of infection via chemotaxis, and will facilitate phagocytosis of pathogens via complement receptor-mediated endocytosis.

As a last note, it is relevant to bring up the presence of ‘natural antibody.’ These (IgM) antibodies are made by a special group of innate (B-1a) B Cells found most prevalently in young organisms and at lower concentrations later in life. Interestingly, these antibodies are produced prior to antigen exposure and have a pre-defined array of specificities including targets such as phosphorylcholine and certain carbohydrates common on bacteria. As such, they are distinctly innate mediators of immunity and are specifically capable of fixing complement on their bacterial targets.

Comparison-of-chemotaxis-of-eosinophils-mediated-by-C3a-and-C5a-Anaphylatoxin-mediated

Comparison of chemotaxis of eosinophils mediated by C3a and C5a.Discipio 1999

 
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Posted by on February 18, 2020 in B-1 B Cells, complement, immunology

 

The Curse of Knowledge

There are several simple problems that are often at the root of communication failures. Sometimes, this may be because the speaker is not thinking clearly. Sometimes this might be because the speaker really does not understand what they are talking about at all. Sometimes, it might even be that the speaker knows what they are talking about too well. In his recent book, The Sense of Style: The Thinking Person’s Guide to Writing in the 21st Century, Harvard Psychologist, Steven Pinker, argues that the curse of knowledge is often to blame. Check out this Inc. article by Glenn Leibowitz on the topic.

 
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Posted by on February 12, 2020 in Uncategorized

 

The Wuhan CoronaVirus

A graphic and a short film on the Wuhan Coronavirus.

The reproduction number is a valuable piece of information that enables epidemiologists to predict how rapidly a virus (or other infectious organism) will spread within a population. For comparison, Measles has a reproduction number of 12-18, which is extraordinarily high, the 1918 influenza that was responsible for infecting some 500 million people worldwide had a reproduction number of 2-3.

The reproduction number tells us nothing about the lethality of the virus, but it does tell us how quickly we can expect it to spread.

Screen Shot 2020-01-30 at 8.57.54 AM

Here is a short film showing the impact that the Wuhan virus is having in one small community in China.

 
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Posted by on January 30, 2020 in Uncategorized

 

Regulating the Regulator of Heat Shock Response in Bacteria

This week in my Molecular class we discussed the role of sigma (σ) factors in regulating transcription. Sigma factors are proteins which bind to the DNA-Dependent RNA polymerase and mediate that enzyme’s binding to the promoters of prokaryotic genes. In this way, we can consider the core polymerase enzyme to be agnostic to which genes are transcribed, it is only the mediation of the sigma factor that guides the polymerase to one gene or another.

Under resting conditions, σ70 is expressed highly and guides the polymerase to constitutively express ‘normal’, housekeeping genes. However, under conditions of stress, such as heat shock, another sigma factor, σ24, becomes active, leading to the transcription (and subsequent translation) of a third factor, σ32. This third sigma factor then leads the polymerase to translate a number of heat shock response proteins such as chaparones, which help prevent protein denaturation and aggregation.

The question is, how does σ24 know when to become active and initiate this heat shock cascade?

Before answering this, we should ask ourselves what to expect.

How can a cell respond to heat shock before the heat shock proteins are made? The answer must be that somehow the heat shock itself leads to a difference in the sigma factors being utilized. I speculated in class that one possible mechanism would be that either the polymerase or σ24 might partially denature in a way that their association was favored over that of the polymerase and σ70.

Although that could work, it turns out not to be the case.

Boldrin et al. examine this question in the bacteria Mycobacterium tuberculosis and suggest that an anti-sigma factor, regulator of sigma E factor A (RseA), binds σ24 (which they call σE – however, I will continue calling it σ24 for consistency) and sequesters it under resting conditions. They continue, “[i]f [RseA] acted as a σ24-specific anti-sigma factor, we would expect to detect an upregulation of those genes whose expression is regulated by σ24 when [RseA] is absent.” One such gene regulated by σ24 is sigB.

To demonstrate that σ24 does regulate sigB, cells missing σ24 were generated. Indeed, the expression of sigB was repressed about 10 times in these cells compared to the wild type (Figure 4). Similarly, when RseA was overexpressed, sigB was also repressed(Figure 5). Genes not regulated by σ24 were unaffected by the deletion of σ24 or the overexpression of RseA (data not shown)Screen Shot 2019-10-18 at 10.13.59 AM.png

Taken together these data indicate that the absence of RseA specifically increases the activity of σ24.Screen Shot 2019-10-17 at 4.35.45 PM.png

De Las Peñas et al. confirm that RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits σ24.

 

 

 

Of course the rabbit hole continues to deepen as we ask how RseA knows to release . It turns out that (cellular) envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS, but that’s as far as we’ll go here. I hope this helps!

Screen Shot 2019-10-18 at 10.14.44 AM.png

 

Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE Francesca Boldrin, Laura Cioetto Mazzabò, Saber Anoosheh, Giorgio Palù, Luc Gaudreau, Riccardo Manganelli & Roberta Provvedi  Scientific Reports volume 9, Article number: 4513 (2019)

The σE‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σE Alejandro De Las Peñas, Lynn Connolly, and Carol A. Gross 31 October 2003

See also:

The Single Extracytoplasmic-Function Sigma Factor of Xylella fastidiosa Is Involved in the Heat Shock Response and Presents an Unusual Regulatory Mechanism José F. da Silva Neto, Tie Koide, Suely L. Gomes, Marilis V. Marques Journal of Bacteriology Dec 2006, 189 (2) 551-560

 
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Posted by on October 18, 2019 in Uncategorized

 

Ocean

I didn’t know the song Ocean, by the Velvet Underground, despite owning Unloaded for years, until I hear a cover version by MGMT off their Late Night Tales album.

I wasn’t very impressed. in fact, as the song neared its end, I was struck by the realization that it had a bizarre ripoff of Alive and Kicking‘s signature riff. I was a little angry that MGMT had pulled off this larceny and was determined to listen more closely to see if I was right.

Unfortunately, it was as if the song didn’t exist – I kept googling “MGMT ocean” to find a youtube version to play side-by-side against the Simple Minds’ song, but I kept coming up short. Until I added the album name, ‘Late Night Tales‘ to my search and found the Underground version. And then I was stuck.

Alive and Kicking came out in 1985,  Ocean came out on the Velvet Underground’s VU album the same year. So, neither was a copy of the other, just two bands hitting similar riffs at the same time. However,  Ocean had been recorded for MGM studio years earlier, in 1969, and was simply shelved until its release on VU.

Except that there was another version re-recorded by the Underground, but released on Lou Reed’s eponymous album in 1972. So it is possible that Simple Minds had heard this and was influenced by it.

Or maybe I’m just hearing things that aren’t there.

And if I’m really interested in hearing an ocean song, it’s probably this one:

 
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Posted by on March 23, 2019 in Uncategorized