Ostermeier lab best universities in india

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.

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. Top universities london 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.

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.

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.

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.

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!

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.

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. Universities in 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.

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. Washington university law second, daprins and, to a lesser extent, monobodies show promise as input domains for a modular platform for the rapid development of switches.

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.

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).