August 3, 2012
Olá from Campinas, Brazil! I can't believe I'm already halfway through my internship at Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), known in English as the Brazilian Bioethanol Laboratory. Time is passing by so quickly that I'm still not completely used to the idea of living and working in Brazil. The real telling of time is how my ears have long since accustomed to hearing Portuguese at every moment. In lab, I'm always learning new things, and my research is simply fascinating. A huge highlight of my internship is the amazing people I'm so fortunate to be working with.
I joined a team of researchers who work on screening metagenomic libraries for biomass conversion enzymes. In Brazil's sugar cane fields, the sugar cane remains naturally degrade in the soil. This suggests that microorganisms are living in the soil that can enzymatically convert the plant biomass into sugars for food. Using this idea, CTBE researchers compiled a metagenomic library by first extracting DNA from soil and inserting DNA pieces into a plasmid vector to transform E. coli. Collections of E.coli colonies, each colony transformed with an unique insert of DNA, make up the metagenomic library. Somewhere in this library lie the hidden treasures we screen for: genes from microorganisms in soil that encodes for biomass conversion enzymes.
Why are enzymes important? Biofuel is generally produced by degrading biomass into sugars, and then using yeast to ferment the sugars into ethanol, the fuel. At the biomass degradation step, a cocktail of enzymes (along with other chemical and/or mechanical treatment) is necessary to break down the complex and recalcitrant biomass. The focus of my research is to discover an efficient enzyme to possibly improve the biomass conversion step, and also to better understand how the soil microbial community degrades biomass in nature.
In addition to metagenomics work, I spent much of two weeks synthesizing Taq polymerase. At first I was not sure why I needed to do this. Although Taq is necessary for DNA amplification and is an enzyme widely used throughout the metagenomic process (i.e. plasmid extraction, sequencing DNA inserts in the plasmid, and amplifying a specific gene once an insert is sequenced), it is much easier and possibly less expensive to buy it. It was my colleague, João, who helped me appreciate that besides Taq's practical uses, making Taq firsthand would give me a deeper understanding of protein expression.
Protein expression is the "what's next" after metagenomic screening detects a gene encoding an enzyme. After finding the gene, we need to perform protein expression to actually produce the enzyme and thus be able to characterize it through assays. Taq is used to amplify the enzyme gene that has been identified and designed specific primers for, and then the gene is inserted into the pET plasmid vector to transform E.coli. Various steps follow to induce expression, i.e. adding the reagent IPTG.
Guess how Taq is made? When the pieces all fell together, I was simply amazed. Just like for the biomass conversion enzyme gene, the Taq gene is also inserted into the pET vector to transform E.coli. The same steps, like adding IPTG, follow. Essentially, Taq is used before the protein expression step, but we use protein expression to create Taq first. SO awesome!
Mini biotech inception besides, did my homemade Taq end up working? I'll save the results and more on the screening process for blog post dois. Last thoughts: SF bay area climate is wonderful but the weather in Campinas has been perfection. I'm loving my sunny Brazilian winter!
August 10, 2012
Last time I left off, I had on hand a cloudy yellow Taq polymerase solution. Taq is normally not yellow, so I was quite nervous about whether this homemade version would work. To test its activity I used Taq with metagenomic soil DNA and DNA from bacillus pumilus. If segments of these two types of DNA could be replicated into high enough amounts to visualize on a gel, my homemade Taq was successfully made. When I finally saw bands in my test gel, it made my day to know that work paid off!
Now armed with working Taq, I can proceed on to metagenomic library screening. While there are many methods to do this, one traditional protocol is growing metagenomic library colonies on plates and adding a substance called congo red. When a colony secretes a biomass degrading enzyme, congo red will allow an orange halo to form around the colony. One disadvantage, however, is that bacteria may not always secrete their enzymes, instead processing the biomass intracellularly. I had the opportunity of assisting my colleague Thabata, when she used congo red to confirm her previous screenings for the enzyme mannanase.
I will actually not be working with congo red but using a strategy that will enable me to complete screening within just a few weeks. To save time the vast metagenomic library was pooled into just six 96 well plates. Each column of a 96 well plate now contains one entire 384 well plate. The novel protein I am screening for is an enzyme called xylanase, which degrades biomass into xylose sugars. My supervisor Fabio calls this procedure "fishing for a new xylanase." But what is the bait for this new xylanase that never been caught before?
As it turns out, we are not starting from zero knowledge about this new enzyme. There are many variations of xylanase, but throughout all these variations there are conserved regions in their DNA sequences that are identical. Degenerate primers that anneal to conserved regions were created, and now I can use these primers to search for a novel xylanase.
The first step is to extract plasmidial DNA from each pooled well through a protocol called mini-prep. After I have verified the DNA yield from the mini-prep, I can perform PCR with the primers to amplify only the plasmids containing a sequence for xylanase. When something has been amplified, presumably the xylanase gene, I will be able to see a band on a gel, and voila! I have my novel enzyme. What follows is an extensive procedure involving going backwards to find out exactly which colony the gene came from, discovering the entire sequence of the gene, and using protein expression to produce this new xylanase Metagenomic research is quite tricky, and this complicated protocol is keeping me on my toes. Like with any other research, it is important to be aware of not only what I am doing (i.e. adding detergent to wells), but what is happening in a molecular sense (lysing cells).
August 13, 2012
It is nearing the end of my stay here in Campinas. I have finally started to become comfortable using some Portuguese, from greeting people with "Oi! Tudo bem?" to simple things like asking the time and buying a coconut from a street vendor. My funny attempts at the language continue to amuse my colleagues, and I in turn love talking to them about various cultural differences. In Portuguese, the "r" is pronounced like an "h", so once when Thabata said and meant "rare enzyme" I heard "hare/hair enzyme", leading to some laughs. In Joao's case, he says a characteristic "not!" whenever he actually means "no." I told Fernanda how to count in Mandarin, and we love to exclaim "gan bei!" (cheers!). Among countless other things, I will really miss these quirks when I return to the states.
I am continuing to work with the 6 plates by screening 2 plates at a time. My favorite part is working with humongous gels that can run up to a hundred lanes. Every time a gel finishes running and I scrutinize it for a band, I almost feel like a lottery player looking for the winning numbers on a ticket! So far, no luck, but we have found some suspicious looking lanes that I will further investigate by taking the corresponding wells through the protocol again, and reexamining the results.