Legoland TALES of Binding DNA by Design

Imagine that you could have a protein that binds to a sequence in a chromosome where you want to activate transcription, nick or break DNA to target insertion or recombination, or create a DNA lesion for screening DNA repair pathway factors…imagine that you could build such proteins very much like putting together legos. Yes, it is possible based on findings about transcription activator-like effector (TALE) proteins. These are plant pathogen transcription factors naturally used to facilitate invasion of host species which have been rekindled to direct DNA binding in other species (1, 2).

Previously, zinc-finger nucleases (ZFNs) have been the focus of genomic modification tool development, but with only limited success. It is not easy to design or select a ZFN using available technologies. In comparison, TALEs have a modular 34 amino acid domain as a basic unit that recognizes a DNA base, with specificity mostly determined by residues 12 and 13. In other words, by using as few as 4 modules with dedicated diamino acids 12 and 13, one can create a protein that binds any DNA sequence.

However, it is not necessarily an easy construct to make because the highly repetitive sequence of TALEs causes plasmid instability during cloning. A team at Harvard recently published a method of minimizing repetitiveness and allowing step-wise ligation; (3). Other aspects of using TALEs involve the designing of an effector domain, e.g. DNAase or transcription activation domain, and packaging the “warhead” in a delivery vehicle such as a lentivirus. The unfolding of the TALEs is just starting, the future seems exciting.

1. J. Boch et al. Science 326, 1509 (2009); published online 29 October 2009
2. M. J. Moscou, and A. J. Bogdanove, Science 326, 1501 (2009)
3. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

New Product of the Week 030711-031411: 60x15mm EcoCulture™ Vented Dishes

Promotion of the week 030711-031411: Lenti-loxP-RFP viruses for creating cells that will have an RFP with loxP sites as a target of Cre. One time 25% off.

Tags: DNA binding, DNA repari, homologues recombination, modular, nuclease, TALE, TALEs, transcription activator, ZFN, ZFNs

New SurfaceBind gDNA Isolation and Purification

Allele Biotech’s SurfaceBind Genomic DNA Pu¬rification Kit is designed for fast, easy, and high-throughput gDNA isolation and purification for lysate obtained through the use of Allele-in-One Mouse Tail Direct Lysis Buffer. Based on our Solid Surface Revers¬ible Binding (SSRB) technology the SurfaceBind system utilizes a plastic tube with its surface coated with proprietary turbo-binders acting to selectively capture and efficiently bind DNA mol¬ecules from reaction mixtures. After lysis of cells, gDNA molecules will specifically interact with the turbo binders and bind to the surface of the tube in the presence of the binding buffer, while pro¬teins and other contaminants will remain in solu¬tion. The DNA can be eluted with as little as 10 microliters of water or buffer for the next application, allowing for a highly concentrated solution.

The entire process of recovery takes less than 10 minutes with only 1 centrifugation step, making it fast and easy. SSRB technology also provides for maxi¬mum DNA capture and release with limited sam¬ple input, without the DNA loss associated with membrane and bead-based technologies.

This is a newly developed product particularly for the Allele Biotech’s customers who use the All-in-One mouse tail genotyping kits: get purified genomic DNA using the same lysate you generated for a quick PCR. The yield and purity will enable direct applications to chip assays, sequencing, Southern blotting, etc.Next time you use Allele-in-One Mouse Tail Direct Lysis Buffer be sure to try our SurfaceBind gDNA Purification kit.

mWasabi-GFP Expression vector with IRES for co-expression. Cat # ABP-FP-WIRES10. email FP@allelebiotech.com for more FP IRES-containing plasmids.

10% off all mTFP1-expressing plasmids this week, check out the vectror you like at shop.allelebiotech.com

Tags: DNA purification, genomic DNA, microarray DNA, mouse tail genotyping, next generation sequencing, one step PCR, Southern Blot

New Frontiers for Research Tool Development in the New Year

Allele Biotech's Green Crystal Ball

Optogenetics
Chosen as the Method of the Year 2010 by Nature Method and mentioned in a number of year-end recaps, this is a technology that allows the use of light to precisely (at least in a temporal sense) control engineered proteins within a targeted cell population. For example, by introducing light-activated channelrhodopsins into neurons, one can use a pulse of light to initiate a movement of ion across the cell membrane. The technology, first reported in 2005 then made headlines as a major impact on neurosciences since 2007, is now being combined with other components in controlling a broader array of biological events, such as DNA binding, enzyme activities, etc. Looking forward, a few areas will be more than likely the frontlines of moving optogenetics into more labs:

Additional combinations: The few known channelrhodopsins and their fast growing variations will be combined with more “effecter” domains to control different events. The challenge will be to find ways to use the structural changes or any responses channelrhodopsins have to stimulating lights in order to trigger a reaction in the associated effecter domain.

Tracking mechanisms: A platter of fluorescent proteins (FPs) will be used as an independent tracking method to follow cells being targeted. FPs that have optical spectra that do not interfere with the optogenetic molecules will be tested and established. In addition, FPs with less toxicity, narrower excitation and emission peaks, and more tolerance to different cellular environment will be preferred and eventually set up as standards.

Delivery tools: To bring the optogenetic reagents into cells like neurons researchers will most likely rely on lentiviral vectors in most cases. Other vehicles such as baculovirus, MMLV-based retrovirus, even herpes virus may find broader applications in this field. Pre-packaged lentiviruses and MMLV-retroviruses already contain optogenetic constructs will become popular products.

VHH Antibodies
The small capture polypeptides based on single-domain Camelid antibodies (nanobodies, nano antbodies or nAbs) and similar VHH domains will become much dramatically more popular this year, judging from the significant increase in demands of the only camelid reagent products, GFP-Trap and RFP-Trap, in 2010. There are a number of NIH initiated programs that aim to find capture reagents that eventually target the complete human proteome. One of the key criteria for the current phase of the relevant NIH Director’s Initiative is ability to co-immunoprecipitate. The Human Proteome Organization (HUPO) recently expressed frustration due to the lack of high quality capture reagents necessary to isolate and identify most proteins. HUPO promotes global research on proteins in order to decode the human proteome. From what we have learned from dozens of publications showing the use of GFP-Trap, VHH molecules pulls down GFP-tagged proteins with unprecedented efficiency and purity. VHH antibodies show strong affinity and specificity, at a level superior or comparable to monoclonal antibodies. In addition, VHH antibodies are increasingly appreciated for their capabilities to recognize concave epitopes by their relatively convex-shaped paratopes. VHH nanobodies are small (~12-15 kD), with a limited number of functionally important disulfide bonds, can be expressed very well in E. coli, and are amazingly stable in extreme denaturing conditions such as heat and acid. They have been shown to be better suited for in vivo and trans-cellular membrane delivery than other antibodies. It should not be surprising that one day in the coming years VHH antibodies will be more dominant than monoclonal antibodies.

Super-Resolution Imaging
One of the goals of developing technologies such as photoactivated localization microscopy (PALM) and related super-resolution imaging (SRI) techniques was to achieve electron microscopy (EM) level resolution without using EM. Now new developments show that maybe combining EM and photoactivable FPs would provide more specific and more detailed morphology. It would be anticipated that more photoconvertible FPs will prove to work well for one type of SRI or another. The event that will bring this technology to nearly every cell biology lab is the improvement and availability of necessary instruments that some companies have already begun to commercialize.

New Product of the Week 010311-010911:

Human let-7b miRNA minigene on lentivirus with RFP reporter, ABP-RP-MILT7BLP

Promotion of the Week 010311-010911:

15% off mWasabi-based organelle markers carried on baculo2mammalian system if order this week (On-Demand products will require about 3-4 weeks for virus packaging after an order is placed). Use code 0103BACFP on fax or email order.

Tags: alpaca antibodies, camelid antibodies, channelrhodopsin, EM, halorhodopsin, optogenetic, PALM, photoactivable FP, photoconvertible FP, STED, STORM, VHH, VHH antibodies

Many Thanks to Give

We would like to thank our customers for choosing Allele’s products and services. The economic conditions have been challenging for the last two years and are still difficult. Allele Biotech could not have possibly achieved what it has within this time period without the support of its customers.

We thank online readers who visit our Blogs, News and Technical Forum to make our webpage one of the highest ranked among biology reagent suppliers; surpassing Clontech, Stratagene, Promega, IDT, etc. (by online ranking service Alexa’s accounts).

We thank our collaborators (some converted from customers) and business partners (manufacturers, distributors, licensors) for working with Allele and making our plan of one new product per week a reality. Allele Biotech has a very innovative and able research team, but working with researchers outside of the company is always a major part of the R&D effort at Allele.

Allele Biotech is obliged to its employees for their creativity, organization, dedication, and professionalism. The culture that they have nurtured here is to be individually motivated as a basic research group, and to be disciplined and organized as a service provider at the same time.

Allele Biotech is a beneficiary of the US government’s policies on supporting biomedical research and innovation. We appreciate the support for our basic research in the forms of grants and contracts from the NIH and the IRS, and ultimately the American tax payers. It is our duty to create and produce better tools for improving human health.

Thank you once again and have a Happy Holiday!

New Product of the Week 112210-112810:

Photoconvertible FP mClavGR (green-to-red) on lentiviral vector pLICO, email FP@allelebiotech.com for product details.

Promotion of the Wee 112210-112810:

10% additional discount off HPLC siRNA’s promotion price, $135/15 nmol, use promo code FB112210si, order by emailing oligo@allelebiotech.com.

Tags: Clontech, collaboration, collaborators, customer opinions, customer service, IDT DNA, IDT oligo, Life Technologies, mClavGR, photoconvertible FP, promega, Stratagene, thanksgiving

Do You Know How Well Your Sunscreen Works?

Skin diseases caused by sun exposure include melanoma, basal cell carcinoma, squamous cell carcinoma, photoaging, as well as sunburn and many other conditions. According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. The vast majority of mutations found in melanoma, according to a 2009 study published in Nature [1], are caused by UV radiation.

Currently, commercial sunscreens are composed of physical sunblocks including zinc oxide and titanium dioxide, and chemical UV (ultraviolet lights) absorbers/filters such as octinoxate for UVB and benzophenone for UVA. The compositions of commercial sunscreen products are disclosed by the manufacturer and regulated by the health product regulatory authorities such the FDA in the US. The UV absorbers/filters are organic chemicals that absorb UV lights within a very limited range of wavelength. Consequently, a combination of different chemicals is needed to achieve “broad-spectrum” protection.

Currently the FDA required test of effectiveness of UV protection measures only UVB, which means there is no way of knowing how effective a sunscreen product is against cancer-causing UVA and damaging visible lights [2]. Even though the life style changes in recent time result in more damaging light exposure such as extended sun bathing on beach or tanning in beauty saloons, etc., only 3 new sunscreen active components (and none of new chemical class) have been introduced to the US market in more than 3 decades. There seems to be a gap between the need and the effort for developing substantially improved skin protection products.

1. Pleasance, E.D., R.K. Cheetham, P.J. Stephens, D.J. McBride, S.J. Humphray, C.D. Greenman, I. Varela, M.L. Lin, G.R. Ordonez, G.R. Bignell, K. Ye, J. Alipaz, M.J. Bauer, D. Beare, A. Butler, R.J. Carter, L. Chen, A.J. Cox, S. Edkins, P.I. Kokko-Gonzales, N.A. Gormley, R.J. Grocock, C.D. Haudenschild, M.M. Hims, T. James, M. Jia, Z. Kingsbury, C. Leroy, J. Marshall, A. Menzies, L.J. Mudie, Z. Ning, T. Royce, O.B. Schulz-Trieglaff, A. Spiridou, L.A. Stebbings, L. Szajkowski, J. Teague, D. Williamson, L. Chin, M.T. Ross, P.J. Campbell, D.R. Bentley, P.A. Futreal, and M.R. Stratton, A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 463(7278): p. 191-6.
2. Botta, C., C. Di Giorgio, A.S. Sabatier, and M. De Meo, Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens. J Photochem Photobiol B, 2008. 91(1): p. 24-34.

New Product of the Week 080910-081510: Drug Resistance: pCHAC-MCS-IRES-NeoR, a new drug-resistant version of Allele’s retroviral vectors. ABP-PVL-IRES10N $325.00

Promotion of the Week 080910-081510: miRNA lentivirus packaging, $300 off listed price. Email vivec@allelebiotechl.com for details, with promotion code V080810.

Tags: fluorescence, light exposure, melanoma, skin cancer, skin care, spectra, spectrum, sunburn, sunscreen, UVA, UVB, visible light