Improved precision, sensitivity, and adaptability of Ordered Two-Template Relay cDNA library preparation for RNA sequencing.

Sequencing RNAs that are biologically processed or degraded to less than ~100 nucleotides typically involves multi-step, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced Ordered Two-Template Relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries.

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9.

An Open Library of Human Kinase Domain Constructs for Automated Bacterial Expression.

Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance.

Production of Purified CasRNPs for Efficacious Genome Editing.

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g.

MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438).

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm.

Background: Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation.

Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community.

Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5-1.

Opsin stability and folding: modulation by phospholipid bicelles.

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-alpha-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin.

The temperature dependence of P680(+) reduction in oxygen-evolving photosystem II.

The temperature dependence for the reduction of the oxidized primary electron donor P680(+) by the redox active tyrosine Y(Z) has been studied in oxygen-evolving photosystem II preparations from spinach. The observed temperature dependence is found to vary markedly with the S-state of the manganese cluster. In the higher oxidation states, S(2) and S(3), sub-microsecond P680(+) reduction exhibits activation energies of about 260 meV.