New paper from Sean on using recombineering for protein engineering

Check out Sean's new paper just published in ACS Synthetic Biology! Here we show that recombineering, a known method of bacterial genome engineering (pioneered first by Court and later by Wang/Isaacs/Church) can also be used for the reliable programmed construction of DNA libraries on plasmids. It turns out this can be a simple way to make libraries of protein expression plasmids for protein engineering studies. We then demonstrate the technique works by engineering a novel thermostable and brighter variant of the small fluorescent protein iLOV. Congrats Sean!

 

 

Check out Rob and Caleb's review on Encapsulins

Check out Rob and Caleb's new review on Encapsulins! In case you haven't heard, encapsulins - also sometimes called nanocompartments - are capsid-like complexes that function as simple organelles in many prokaryotes. These fascinating proteins have only recently been discovered and Caleb and Rob's review is one of the first comprehensive looks at just how diverse the structure and function of this family is.  

Nichols RJ, Cassidy-Amstutz C, Chaijarasphong T, Savage DF. Encapsulins: molecular biology of the shell. Crit. Rev. Biochem. Mol. Biol. 2017 Jun 21;:1–12. 

Graphical abstract FINAL color.jpg

Emeric and Jack join the Savage Lab

A hearty salutations to Emeric Charles and Jack Desmarais, who have joined the Savage Lab from the MCB graduate program. Welcome aboard guys! 

Doctors everywhere!

A huge congratulations to Caleb, Rachel, and Ben on their hooding and official granting of their PhD! Great work everyone!

calebRachelBenGraduation2017.jpg

Dana, Benz, Stacy, and Rayka: You will be missed!

A huge goodbye to Dana, Benz, Stacy, and Rayka who are all moving on this winter to their next adventures beyond the Savage Lab. Thanks again for all of your hard work getting the lab up and running and best of luck with your next steps. Onwards and upwards! 

New insights into the CO2 Concentrating Mechanism

Congratulations to Avi on his paper that was recently published in PNAS! In this paper, done in collaboration with Niall Mangan at UW and Ron Milo at the Weizmann Institute, we develop a sophisticated and complete treatment of the critical reactions of the CO2 Concentrating Mechanism found in cyanobacteria. Surprisingly, we find that pH plays a previously unappreciated role in trapping CO2 in the cell, much in the same way that glucose is phosphorylated upon entry into a human cell. Without a critical adjustment of the pH during the light reactions of photosynthesis, the cell leaks carbon and the process is inefficient. Thus, the work demarcates an important physiological constraint that shapes the efficiency of the system and one to take into account as we think about novel ways of improving photosynthesis. 

Article can be found: here

Code is available here: here

Overview of the CCM modeled in Mangan et al.

Overview of the CCM modeled in Mangan et al.

Rachel's paper on the cyanobacterial stringent response is out!

 A big congratulations to Rachel for her work that appears in PNAS today! Rachel found that the stringent response, a starvation response mediated by the nucleotide ppGpp, helps regulate the physiological transition of cyanobacteria when they are switched from the light to the dark. This paper ties together a number of historical works that noted rapid physiological changes when cyanobacteria experience the dark. Most interestingly, it gives us a new pathway and numerous genes downstream of ppGpp that likely influence how a photosynthetic cell uniquely adapts to the rapidly changing conditions found in its environment.  

Citation: The stringent response regulates adaptation to darkness in the cyanobacterium Synechococcus elongatus. Rachel D. Hood, Sean A. Higgins, Avi Flamholz, Robert J. Nichols, and David F. Savage. PNAS 2016 ; published ahead of print August 2, 2016, doi:10.1073/pnas.1524915113

Model for the cyanobacterial stringent response adapted from Hood et al. 2016

Model for the cyanobacterial stringent response adapted from Hood et al. 2016

New paper on biosensors!

Congrats to Dana and Stacy on their paper describing a method for making metabolite biosensors! Inspired by previous work on recombination and domain insertion by the Lim, Dueber, Ostermeier, Silberg, and Jones groups, Dana and Stacy developed an engineered transposon that allows for rapid shuffling of protein domain relative to one another. Combining this approach with high-throughput assays and next-gen DNA sequencing, they show this method, which we call Domain-Insertion Profiling aka DIP-seq, is a great strategy for making metabolite biosensors fashioned around GFP. We think these type of biosensors have great promise as a tool for imaging and screening metabolites. The paper is available here: 

Nadler DC*, Morgan SA*, Flamholz A, Kortright K, Savage DF. 2016. Rapid Construction of Metabolite Biosensors. Nature Communications 10.1038/ncomms12266. *denotes co-first authors

If you're interested in trying our protocol, please check out the Materials and Methods section. We put a lot of work in making the details transparent and, of course, all of our plasmids are on addgene. 

 

 

 

New paper on encapsulins

A huge congrats to Caleb on his new paper, "Identification of a minimal peptide tag for in vivo and in vitro loading of encapsulin," that was just published online in Biochemistry. Our lab is interested in the ability and effect of encapsulating proteins and other molecules inside protein compartments. Here, building off of beautiful work from Sutter et al., Caleb demonstrates that proteins can be loaded inside the minimal protein compartment from T. maritima known as encapsulin. Importantly, this work identifies a minimal targeting sequence, several different biochemical approaches for doing the loading, and a general strategy for obtaining large amounts of material. 

Identification of a minimal peptide tag for in vivo and in vitro loading of encapsulin. Caleb Cassidy-Amstutz, Luke M Oltrogge, Catherine C Going, Antony Lee, Poh K Teng, David Quintanilla, Alexandra East-Seletsky, Evan R Williams, and David F Savage. Biochemistry. DOI: 10.1021/acs.biochem.6b00294

New paper on engineering allosteric control of Cas9 in Nature Biotechnology

A big congrats to Ben et al. on their paper dropping today in Nature Biotechnology! This work uses randomized domain insertion to explore the 'malleability' of Cas9 for protein fusions. Surprisingly, Cas9 is a robust structure that can accept insertions throughout the protein and remain functional, even as surprising positions such as the middle of alpha helices, etc. Taking this one step further, Ben use this trick to isolate a novel Cas9-estrogen receptor ligand binding domain fusion that is allosterically regulated by small molecule. This system therefore represents a simplified tool for controlling Cas9 activity with a well-characterized ligand. If you're interested in finding out more check out:

Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, Savage DF. 2016. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nat Biotechnol.

Figure 1 from Oakes et al. showing the malleability of Cas9 to domain insertion.

Figure 1 from Oakes et al. showing the malleability of Cas9 to domain insertion.

Rob passes his PhD qualifying exam!

Congratulations to Rob Nichols, a 2nd year PhD student in the MCB program, who just passed his exam. You're now 'qualified!' We hope you enjoyed the PhD thesis simulator too!

New paper from Benz on the carboxysome

A big congratulation to Benz on his recent paper in JMB that solves an old question about the carboxysome! The alpha-carboxysome, which is formed from at least 10 different proteins, possesses an odd, intrinsically disordered protein named CosS2 that recent work suggests (Cai, F. et al., 2015. Life, 5(2), pp.1141–1171.) is critical for carboxysome assembly. Work on CsoS2 has been hampered by strange behavior of CsoS2 and the fact that its single gene leads to the production of two protein gene products, CsoS2A and CsoS2B. Benz used a combination of molecular biology and biochemistry to identify a programmed ribosomal frameshifting site in the mRNA of csos2. This regulatory element programs the ribosome to make two different polypeptides and therefore create two populations of CsoS2 proteins. It is clear there are functional differences between the two isoforms and, towards future work, Benz is curious about how and why these differences occur.

A model for CsoS2 function (yellow) in the assembly of the carboxysome. 

A model for CsoS2 function (yellow) in the assembly of the carboxysome. 

 

The reference for the work is:

Chaijarasphong T, Nichols RJ, Kortright KE, Nixon CF, Teng PK, Oltrogge LM, Savage DF. 2016. Programmed Ribosomal Frameshifting Mediates Expression of the α-Carboxysome. J Mol Biol 428: 153–164. 

Welcome Luke!

A long overdue welcome to Luke Oltrogge, who joined the lab early in the summer! Luke recently received his PhD in Chemistry from Stanford for his work in the Boxer Lab. Luke is interested in the assembly mechanisms of bacterial microcompartments. 

Ben passes his QE!

And then there were three! A big congrats to Ben Oakes, who passed his PhD qualifying exam with flying colors!

Welcome aboard Rob!

Rob Nichols, a first year student from the MCB graduate program, has decided to join the lab. Rob will work on the basic science of cyanobacteria regulate and catalyze carbon assimilation. 

Avi passes his QE too!

A big congratulations to Avi, who passed his PhD qualifying exam today! Onwards and upwards!

Sean passes his Qualifying Exam!

A huge congrats to Sean, who passed his PhD qualifying exam with flying colors! Here is Caleb's call of the exam:

Though his committee tried to quench his excited state, Sean's LOV engineered a glowing response!


A goodbye party for Poh

Best wishes to Poh as she makes the leap from academics into industry! Poh will be moving to Solar Junction, a leader in high-efficiency solar cell production, where she will work in their intellectual property group. Thanks for all of your hard work Poh. You will be missed!