PCR Update: Pipette, Cry, Repeat

Hey everyone, 2019 has been quite a year in terms of ongoing research at Escarpment Labs. The R&D Department has been drumming up some exciting research on all of our yeast strains to better characterize their performances through a massive fermentation project - so stay tuned for some more fun blog posts in the near future on that!

 

In the past few months, I have been working toward optimizing PCR protocols to help us better understand the genetic makeup of our yeast and bacteria strains. This blog post will focus on some of the PCR projects we are currently working on, as well as future PCR projects we are planning. Traditionally, I like to include pug GIFs in my blog posts but since the internet hates me and I’ve used them all, we will have to stick with French Bulldogs this time (not mad though).  

Let’s get started (Okay, I am fine with this change).

What is PCR? 

In a previous blog post titled Demystifying Diastaticus Part 1, I described the basis of Polymerase Chain Reaction, better known as PCR, as well as the various PCR technologies available and their applications.

To recap:  

PCR is a powerful molecular tool capable of amplifying and detecting the presence of specific regions of DNA. DNA can be thought of as the set of instructions or code found inside of every cell that is required for the organism to live, develop and reproduce. This set of instructions is then read, or translated, in the cell to produce proteins that then carry out specific functions within the organism. Each organism has a different genetic code, or different sets of instructions, and this is true across different strains of yeast and bacteria. By using molecular techniques such as PCR, testing for genetic differences in DNA among organisms is possible.

Before any PCR experiments can be run, DNA from your target organism needs to be extracted and purified. This involves lysing or breaking open the cell walls to release the DNA. In doing this, proteins, debris, and other unwanted cell components are also released so the DNA needs to be purified. This process is typically done with a DNA extraction protocol or commercial kits. We are big fans of the Instagene from BioRad to accomplish this. It is an easy, non-toxic, purification kit that can be used on a multitude of organisms and works well for colony PCR. Once the DNA is successfully extracted and confirmed through established PCR protocols, you are able to run your trial experiments.  

NOTE: The process of optimizing the PCR protocol and ensuring your DNA extraction is working as intended is most of the battle and is not as easy as it sounds. It can take a while to get a PCR experiment to work, but once it does it is very rewarding. For the beer folks out there, getting a DNA extraction and PCR working properly is similar to dialling in a crispy Pilsner… There are a lot of variables that can get in the way of success. 

Very accurate depiction of the struggle that is PCR. 

EXTRA FUN SIDE NOTE: DNA extraction on cells this small is not visible to the human eye. But here is a fun experiment you can try at home that explains the process of DNA extraction in better detail and is an extraction you can see. It involves lysing the cells of banana, which then become stringy and gooey! 

Editor's note: in addition to looking up dog gifs, we also do real science at Escarpment Labs. See below.  

Diastaticus STA1 promoter identification  

When I wrote the last PCR blog post, I was developing a screening method for diastatic yeast by amplifying STA1, the gene responsible for producing glucoamylase in diastatic yeast that allows them to consume starches and dextrins to produce dryer beers. This has been greatly useful for us in characterizing all of our yeasts for this gene to improve our yeast handling practices. Now, it is also possible to determine whether a yeast with STA1 will be an aggressive attenuator or not, since it was discovered that some strains are missing a chunk of the gene promoter. Part of this paper was inspired by conversations between Richard Preiss from our team and Kristoffer Krogerus from VTT in Finland, the lead author of the study. We even sent some of our strains to VTT to be included in the study.

When there is a deletion in the STA1 promoter, STA1-positive yeasts grow slower in beer. Fig. 1b from Krogerus et al. (2019). 

 

Myself and Shelby, one of our Co-Op students, are working on identifying the STA1 promoter deletion in our STA1+ yeast. In-house, we have a PCR protocol to accurately test for the presence or absence of this gene (we do offer this service for diastaticus screening - FYI). However, just because the organism possesses the STA1 gene does not mean that gene expression is active and the yeast is producing glucoamylase. If this gene is missing a section of the promoter (a sequence upstream of the gene that allows the ribosome to attach and “read” the gene to produce the protein), glucoamylase will be created at a much slower rate - more than 100x less gene expression!  

 When the STA1 promoter sequence has a deletion, gene expression is massively reduced. Figure 3e from Krogerus et al. (2019). 

 

It's possible to run PCR for both STA1 and the STA1 promoter activation sequence at the same time, called multiplexing. This gives you more data from the same amount of PCR effort. Win! Figure 2c from Krogerus et al. (2019).  

We can now combine our STA1 protocol with a primer set to amplify the promoter region during the same reaction (this is called multiplexing). This should result in a PCR gel showing two bands if the promoter and gene both exist in the target genome (implying aggressive diastatic activity) and only one band if the STA1 gene is present but the promoter sequence is not (implying less diastatic activity). This means it is possible to characterize diastatic yeasts found as contaminants much faster.

 

Why is all of this important? It's important because until now it's been really tricky to figure out how much risk a particular STA1-positive yeast poses. 

While growth on LCSM agar often relates to diastatic activity, it is an indirect test that simply screens for copper sulfate resistance. Starch agar plates accurately show diastatic ability but can take up to three weeks to see a colour change. With the PCR for the STA1 promoter, we can predict whether yeast is an aggressive diastatic strain or not, in the span of hours instead of weeks. 
 

We have now screened our known diastaticus strains for the STA1 upstream activation sequence, and are happy to report the following:

UAS-positive: French Saison, Spooky Saison, Old World Saison Blend, Dry Belgian Ale
 

UAS-negative: Cerberus, Wild Thing, Ontario Farmhouse, New World Saison, St. Lucifer, Saison II 
 

These results line up quite nicely with the previous work we did with starch agar plates, but now we can get results from a yeast colony in a few hours instead of a few weeks! Yay, science!

Bacterial Identification

Over the past 8 months, I have been chipping away at a protocol for detecting bacterial contaminants via PCR and sequencing. The protocol had to be robust enough to detect any common bacterial contamination seen in brewing while being specific enough to identify the organism later when sequenced.

The process began by testing primers that would amplify a portion of the bacterial 16s ribosome that is conserved in all bacteria but with some variability. This sequence differs enough between most species allowing for the genetic sequence to vary and for species to be differentiated from one another. To do this, we used a pair of 16s-specific primers. After a few failed PCRs, we swapped out the primers and protocol with a new set to amplify the same target gene, this time using a V6r/V3kl primer set (Carlucci et al. (2019) - thanks Christian).   

 

While the protocol worked for many bacterial contaminants saved in our library, some Gram-positive bacteria such as Lactobacillus proved to be difficult to extract DNA from following our usual protocol (using Instagene from BioRad). Gram-positive bacteria have a thicker cell wall than Gram-negative bacteria which is composed of peptidoglycan that reinforces the cell wall. This can make breaking the cell open difficult. Confusingly, it seemed like some Lactobacillus in our library extracted without a problem, while others resisted our efforts.

 

After many DNA extraction experiments, which involved longer vortex steps, boiling samples, and chemical additions, a solution was finally found that could consistently lyse these Gram-positive cells. The use of glass beads in combination with vigorous vortexing was successful. The DNA extraction, in combination with the V6r/V3kl primer, set used in the PCR has proven to be a successful method for amplifying the target gene on the 16s ribosome for every brewery contaminant and tasty Lacto strain we’ve thrown at it, which allows us to successfully sequence the 16s fragment and match to the target organism.

 

If you're working in a brewing lab and looking for more economical ways to get DNA out of lactic acid bacteria, drop us a line.  

After a few trip-ups, you eventually get there. :)

 

Having this PCR protocol in house, in combination with our internal protocol for yeast sequence identification allows us to do some pretty cool things. We are now not only able to plate samples and identify all contaminants based on gene sequence, we can also perform wild captures with more confidence. This means we can collect multiple samples from nature (or clients can), and isolate as many organisms as possible from the source material. We can then screen each yeast and/or bacteria to select for target organisms (ex. Saccharomyces cerevisiae and/or Lactobacillus rather than unwanted cultures). From this information, we can run small scale ferments and identify new potential products based on predicted performance - in much less time than if we were unable to identify the organism and had to test all collected cultures through more labour-intensive methods. What this means is if you’re looking for your very own wild yeast or bacterial strain to make truly unique beers, this is becoming an easier and higher quality process through Escarpment Labs. 

Yeast Fingerprinting Projects 

Capturing samples and fingerprinting them is sort of like an episode of Maury where you are trying to determine who the father is. Using a special PCR that amplifies variable parts of the yeast genome, multiple bands are produced and can be compared to known strains (Legras and Karst 2013). For a fun project to train on PCR, Chris Saunders from the lab team isolated yeast from a can of his favourite hazy beer and tried to identify the parent strains suggested to be used in the beer (he was successful in finding 1 out of 3 strains, which was cool!).

Results from the PCR Fingerprinting “Paternity Test”. Chris was able to identify WB06 as one of the yeasts by comparing the banding patterns, and was able to identify that two yeast he thought were different (E1 and H1) during the isolation process were the same!  

We can also use PCR fingerprinting as a confirmation tool when we’re making new hybrid yeasts. More on that in a few months ;-) 

Future Projects

Some other fun PCR projects are on the horizon or in the initial phases of testing that we are hoping to complete by Christmas if all goes well. One of these projects is to screen our Lactobacillus library for the HorA gene, responsible for conferring hop-resistance to the organism allowing us to identify which strains can be used in hopped wort. This would also be valuable when screening new Lactobacillus strains for product development, such as mixed culture souring blends (we do have more on the way). We think that most of the mixed culture souring blends on the market are not yet optimal and that there is room for improvement and innovation in this area. Research can get us to the next level! 

Other PCR projects include using specific primer sets to quickly identify Saccharomyces species, including Saccharomyces paradoxus, Saccharomyces eubayanus and Saccharomyces bayanus. Surprisingly, hybrid yeasts pop up a lot in the brewing world. While it is well known that lager yeast are a hybrid of Saccharomyces cerevisiae with Saccharomyces eubayanus, it’s also been rumoured that some Belgian ale strains are interspecies hybrids, and sometimes we find wild yeasts that appear to be hybrids as well.

For context, S. paradoxus is a non-conventional brewers yeast strain that is rumoured to be the reason behind the performance of popular Belgian strains. Interestingly, we find this yeast frequently on and around trees up here in Canada! S. eubayanus genetics convey cold and stress tolerance not found in other yeast. Lager strains are typically hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus, inheriting their affinity for malt sugars from the S. cerevisiae parent and cold tolerance from the S. eubayanus parent, respectively. Lastly, S. bayanus strains are similar to S. cerevisiae strains, but are typically used in wine production rather than beer. Being able to classify strains in our collection into these categories will allow us to do some cool yeast breeding projects to create hybrid yeasts with new fermentation capabilities. It will also allow us to quickly identify Saccharomyces yeasts from wild isolation projects.  

As you can see, there are many PCR projects ongoing at Escarpment Labs. Although I could talk for hours on each mentioned, I hope this provides some insight into the amount of effort, work, and real science we put into each of our products. The driving force behind all of this is to ensure our products maintain the highest level of quality and integrity, at all stages of the production process starting with initial R&D testing. 

 

Alex Mitro is a big fan of pugs and is Project Coordinator at Escarpment Laboratories. At Escarpment Labs, he is responsible for developing international export plans, regulatory compliance, and spearheading various external and internal quality control (QC) jobs. One of the QC projects he is currently involved with is to optimize methods for screening contaminants using PCR, as well as R&D efforts to identify and test yeasts and bacteria in our strain collection. 

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References:  
Carlucci, C. et al. Effects of defined gut microbial ecosystem components on virulence determinants of Clostridioides difficile. Scientific Reports 9, p885 (2019).  
Krogerus, K., Magalhães, F., Kuivanen, J., and Gibson, B. (2019). A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains. Appl. Microbiol. Biotechnol. doi:10.1007/s00253-019-10021-y.
Legras, J.L., and Karst, F. (2003). Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation. FEMS Microbiol. Lett. 221, 249–255. doi:10.1016/S0378-1097(03)00205-2.
Pengelly, R. J., and Wheals, A. E. (2013). Rapid identification of Saccharomyces eubayanus and its hybrids. FEMS Yeast Res. 13, 156–161. doi:10.1111/1567-1364.12018.