Two new NSF grants
June 28, 2018
The Ostermeier lab received two new NSF grants. The aim of the first (from NSF-CBET) is to develop modular protein switches. The aim of the second (from NSF-MCB) is to study underexplored mechanisms that constrain the evolution of proteins.

Congratulations Dr. Xiong!
November 2nd, 2017
Earlier this semester, Tina Xiong successfully defended her thesis on using CRISPR/Cas9 to target DNA methylation of CpG sites. A large portion of her thesis work was published in July in Scientific Reports, with a follow up paper soon to be submitted. Tina is now off to a Senior Scientist position at Merck.

RNA-programmed DNA methylation using CRISPR/Cas9
July 27, 2017
Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by the level of DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation.

Today, Scientific Reports published Tina Xiong's paper describing our use of CRISPR/Cas9 to target methylation. This work is a collaboration between our lab, the Winston Timp lab (JHU), and the Carl Novina lab (Dana-Farber Cancer Institute) including Ostermeier lab alumn Glenna Meister. We describe a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site based on guide RNA sequences. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing guide RNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.


Tiana Warren receives the 2017 JHU Diversity Recognition Award
May 18, 2017
Congratulations to Tiana Warren for receiving a 2017 JHU Diversity Recognition Award! Tiana has had leadership roles in several organization with the Whiting School of Engineering, most notably the Black Graduate Student Association, the National Organization for the Professional Advancement of Black Chemists and Chemical Engineers, and the Johns Hopkins Diversity Leadership Council. As a KSAS/WSE Graduate Diversity Fellow she has advised divisional leadership on issues related to graduate student diversity and inspired the production of a Homewood-based, graduate URM resource and community handbook.


Elad Firnberg on NPR's Marketplace talking about Revolve
September 30th, 2016
NPR's Marketplace talked to Ostermeier lab alumnus Elad Firnberg yesterday about how free undergrad tuition at Cooper Union enabled him to co-found Revolve Biotechnologies. Revolve Biotechnologies is a life science company focused on engineering better proteins using novel directed evolution technologies. The company's IP portfolio was developed in our lab. In addition to its internal protein pipeline, Revolve is currently offering a service for construction of custom DNA variant libraries.


Congratulations Dr. Shelat!
September 27th, 2016
Earlier this month, Nirav Shelat successfully defended his thesis on engineering the herpes simplex virus thymidine kinase for enzyme prodrug therapy. PLos ONE just published his paper on the development of a new positive genetic selection for nucleoside kinase activity in E. coli.

Tina Xiong wins poster award at SEED!
July 29th, 2016
Congratulations to Tina Xiong for winning a Biotechnology Journal-sponsored poster award at the 2016 Synthetic Biology: Engineering, Evolution & Design (SEED) conference in Chicago last week for her poster entitled "RNA-programmed DNA methylation". Tina earned one of only six poster awards, and there were 234 posters!


Survey of epistasis along an adaptive pathway
June 10th, 2016
An understanding of the role of intragenic epistasis and tradeoffs in adaptation is central to an understanding of protein evolution. Today, Journal of Molecular Biology published Barrett's extensive and systematic study of a series of fitness and epistatic landscapes along an adaptive pathway (Shifting fitness and epistatic landscapes reflect trade-offs along an evolutionary pathway J. Mol. Biol. 428, 2730–2743), the first study of its kind. He measured the effect on ampicillin resistance of ~12,500 unique mutants of alleles of TEM-1 β-lactamase along an adaptive path in the evolution of cefotaxime resistance. This series of fitness and epistatic landscapes provided extensive experimental insight into the relationships between mutation, protein structure, protein stability, and epistasis and revealed the tradeoffs inherent in the evolution of new functions. We found pervasive epistasis involving adaptive mutations that can be partially understood in terms of protein structural and stability considerations. Adaptation moved the protein to a more rugged and precarious region of the fitness landscape, which is a cost of adaptation.


When bad is good
January 29, 2016
Barrett's paper Environmental changes bridge evolutionary valleys (Sci. Adv. 2(1):e1500921) was highlighted in "This Week In Science" in today's issue of Science. The paper demonstrates that evolution through inferior intermediates can paradoxicaly lead to superior outcomes.

Barrett subjected TEM-15 beta-lactamase to four different selection regimen, three of which involved environmental changes that alter the fitness landscape. The "negative selection" regimen (which included steps for selection of mutations that substantially decrease antibiotic resistance) outperformed the other strategies and ultimately resulted in proteins conferring the highest resistance. In the analysis of one such high resistance protein, he found that an initial, severely deleterious mutation is an initial gateway to a relatively inaccessible area of sequence space. Furthermore, this mutation participates in higher-order positive epistasis with a number of beneficial mutations, compensating for the increasing negative epistasis between beneficial mutations as they accumulate (i.e. compensating for diminishing returns). The ability of "negative" selection and environmental changes to provide access to novel fitness peaks has important implications for natural evolutionary mechanisms as well as applied directed evolution.


Modular protein switches built from antibody mimetics
December 7, 2015
In a just published paper in Protein Engineering Design and Selection, we demonstrate that protein switches can be built using DARPins and monobodies as input domains. The DTRA and NIH funded research was initiated by Manu Kanwar and Amol Date and fully realized by first author Nate Nicholes (with some assistance from Pamphile Beaujean and Pricila Hauk).

Our manuscript describes how domain insertion can be used to create protein switches that consist of fusions between antibody mimetic proteins (monobodies and DARPins) and the enzyme beta-lactamase. These switches' enzyme activity is modulated by the antibody mimetic domain’s ligand. We demonstrate that these switches exhibit modularity in the sense that new switches can be created simply through the introduction of mutations in the antibody mimetic domain that are known to cause binding to the new ligand. There are two significant conclusions from our work. First, it is possible to create protein switches by domain insertion using input domains that do not undergo large conformational changes upon ligand binding. Second, DAPRins and, to a lesser extent, monobodies show promise as input domains for a modular platform for the rapid development of switches.


Two recent protein switch papers
October 13, 2015
Two of our papers on protein switches have recently appeared in Biotechnology and Bioengineering.

In the first paper, Jay Choi together with Maya Zayats from Peter Searson's lab show how an electrochemical signal can be used as an exogenous input to control protein switch function via reduction of the engineered disulfide bonds. This study suggests that a disulfide-containing protein switch is a potentially useful platform for bioelectronic sensors with remote control of the sensing ability.

In the second paper, Nate Nicholes and Jen Tullman led an effort to create switches by fusing TEM-1 beta-lactamase and a variety of paralogous periplasmic binding proteins. The results indicate that the emergence of the switch property likely depends on the precise molecular details of the fusions and cannot be easily predicted from some overall general structural property of the fusion topology.


New NSF grant on targeted methylation
June 15, 2015
NSF-CBET has funded a joint proposal betweeen our lab and Carl Novina's lab at the Dana-Farber Cancer Institute. The project seeks to develop technology for targeted DNA methylation in human cells.

Designing multi-input protein switches based on the conformational ensemble model of allostery
April 22, 2015
Jay and Abby's work on the semi-rational design of multi-input protein switches was published today in Nature Communications. The paper demonstrates how allostery can be established in a non-allosteric fusion protein (between maltose binding protein and TEM1 β-lactamase) through the introduction of mutations designed to increase the conformational entropy of the β-lactamase domain. Biophysical studies support our hypothesis. Our work adds to the growing appreciation of how intrinsically disordered regions can contribute to protein function and lends further support to the conformational ensemble model of allostery. The work was a collaboration with Vince Hilser (JHU).


Improved protein switches with therapeutic potential
November 26, 2014
Clay and Arjun's work on improving our cytosine deaminase switches for use in combination with prodrug therapy is published in PLoS One.

Biotechniques features our targeted methylation research
October 8, 2014
The September issue of Biotechniques has an article on targeting DNA methylation to the genome that features Brian's recent paper in PLoS One.


Jay Choi's paper on protein logic gates published
August 21, 2014
Today ACS Synthetic Biology published Jay Choi's paper on the rational design of protein switches to function as logic gates. The paper describes an alternative approach to gene circuits for introducing logic control elements into cells.


Another new NSF grant
June 17, 2014
More good news from NSF. The lab received a grant from the NSF-DEB Evolutionary Genetics Program to study epistasis.

Brian's paper on improved targeted methyltransferases published
May 8, 2014
Brian's paper on the creation of highly specific DNA methyltransrerases was published today in PLoS One.

New NSF grant on directed evolution
April 21, 2014
The lab received a new grant from the NSF-CBET Biotechnology, Biochemical, and Biomass Engineering Program to explore alternative pathways in directed evolution.

Tiana Warren offered two fellowships
April 18, 2014
Congratulations to Tiana Warren for being selected to receive both an NSF Predoctoral Fellowship and a Ford Foundation Fellowship. Tiana plans to accept the NSF Fellowship.

Marc Ostermeier elected to AIMBE
March 24, 2014
Marc Ostermeier was elected as a Fellow in the American Institute for Medical and Biological Engineering.

Dr. Clay Wright!
March 14, 2014
Congratulations to Clay Wright for successfully defending his thesis today. Next up for Clay (besides getting married) is a postdoc at The University of Washington with Jennifer Nemhauser in the Department of Biology.

Elad's fitness landscape paper published
February 23, 2014
Elad's paper describing a comprehensive map of the mutational landscape of the TEM-1 β-lactamase gene was published online today in Molecular Biology and Evolution.

Dr. Brian Chaikind!
January 17, 2014
Congratulations to Brian Chaikind for successfully defending his thesis today. Next up for Brian is a postdoc at Harvard with David Liu.


T. Xiong*, G. E. Meister*, R. E. Workman, N. C. Kato, M. J. Spellberg, T. Fulya, W. Timp, M. Ostermeier, C. D. Novina
Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
Sci. Rep., 7: 6732 (2017) [full text]

L. F. Ribeiro, T. D. Warren, M. Ostermeier
Construction of protein switches by domain insertion and directed evolution
Methods Mol. Biol., 1596, 43-55 (2017)

N. Y. Shelat, S. Parhi, M. Ostermeier
Development of a cancer-marker activated enzymatic switch from the herpes simplex virus thymidine kinase
Protein Eng. Des. Sel., 30, 95-103 (2017) [full text]

N. Y. Shelat, S. Parhi, M. Ostermeier
A positive selection for nucleoside kinases in E. coli
PLoS One, 11(9):e0162921 (2016) [full text]

J. H. Choi, T. Xiong, M. Ostermeier
The interplay between effector binding and allostery in an engineered protein switch
Protein Sci., 25, 1605–1616 (2016) [full text]

B. Steinberg, M. Ostermeier
Shifting fitness and epistatic landscapes reflect tradeoffs along an evolutionary pathway
J. Mol. Biol., 428, 2730–2743 (2016) [full text]

L. F. Ribeiro, J. Tullman, N. Nicholes, S. R. B. Silva, D. S. Vieira, M. Ostermeier, R. J. Ward
A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination
Biotechnol. Biofuels, 9:119 (2016) [full text]

B. Steinberg, M. Ostermeier
Environmental changes bridge evolutionary valleys
Sci. Adv., 2(1):e1500921 (2016) [full text]

N. Nicholes, A. Date, P. Beaujean, P. Hauk, M. Kanwar, M. Ostermeier
Modular protein switches derived from antibody mimetic proteins
Protein Eng. Des. Sel., 29 77-85 (2016) [full text]

J. Tullman*, N. Nicholes*, M. R. Dumont, L. F. Ribeiro, M. Ostermeier
Enzymatic protein switches built from paralogous input domains
Biotechnol. Bioeng. 113, 852-858 (2016) [full text]

J. H. Choi*, M. Zayats*, P. C. Searson, M. Ostermeier
Electrochemical activation of engineered protein switches
Biotechnol. Bioeng., 113, 453-456 (2016) [full text]

J. H. Choi, A. H. Laurent, V. J. Hilser, M. Ostermeier
Design of protein switches based on an ensemble model of allostery
Nat. Commun., 6:6968 (2015) [full text]

        Mutational tolerance in TEM-1 β-lactamase.