Solving the world’s problems with new biotechnology

The ability to isolate, create, synthesize, or artificially evolve living organisms towards desirable phenotypes may be increasingly important for solving many of the problems the world is facing. Such problems may include creating renewable energy using biowaste, finding biocontrol products that kill food-spoiling fungi “organically”, or assaying pathogens in the field using synthetic biological detection systems. With the arrival of synthetic biology, “it is possible to design and assemble chromosomes, genes and gene pathways, and even whole genomes”, according to the J. Craig Venter Institute. That is, if you know which genes or gene pathways you would need to put into the synthetic genome that would lead to the desired traits. So far, most published synthetic biology work involves bringing in transcription factors from a non-host source to set up an artificial network like circadian oscillators, showing that it can be done and it is interesting.

Through the process of evolution biological systems aptly self-engineer favorable traits in order to survive, but these changes require millions of years to manifest. However, there are quicker adaptations to environmental cues, such as developing antibiotic resistance, which can be achieved through a small number of mutations in hundreds or even dozens of generations. The question is how to harness this kind of adaptation for new strains that can be used as products with defined purposes? As a first requirement, you must have an assay for identifying the wanted mutants or method for augmenting their subpopulation, which is not necessarily easy and normally takes some clever designs to establish. Since evolutionary success in nature results from continuous “rounds” of gene mutagenesis, expression and selection, an evolution in the lab should ideally proceed with continuity. Previously, each round of mutation and selection takes a few days to complete. Recently, Esvelt et al. in David Liu’s lab at Harvard demonstrated one way of doing in vitro continuous evolution, by creating a lagoon of mixed E. coli and phages. By continuous dilution of the phage population through outflow, those phages that remain in the pool with properties that help them propagate in the host bacteria will have a better chance to regenerate and accumulate mutations towards the design of the assay [1].

Another aspect of natural evolution is that it occurs in a heterogeneous environment separated into niches of subpopulations with uneven stress levels. Although most evolutions with human intervention were conducted in a homologous population under the same stress and selection, a spatially complex environment may speed up evolution. This may not be easy to imagine, but if a mutant acquires some level of resistance to its environmental stress level and has a chance to move to join a population under higher stress, its relative fitness will likely increase. In addition, in a smaller population in the niche under higher stress, the mutant with marginally beneficial properties acquired under lower pressure can take over more quickly. This was demonstrated by Zhang et al. who showed that with a gradient of antibiotics applied to an array of microwells interconnected through tiny channels, new resistant strains can evolve in less than a day. Without the gradient, or separate the interconnected niches into discrete wells, no resistant populations could be obtained [2].

With more understandings like these and equipped with large scale gene synthesis, chromosome assembly, and deep sequencing technologies, we should see increasing numbers of human-made organisms serving special needs for food, health, energy, and the environment. Synthetic biology or artificial evolution won’t solve all the world’s problems, but if applied effectively and diligently, they can certainly help with many critical aspects as the technology “coevolves” with the environment.

[1] Kevin M. Esvelt, Jacob C. Carlson, & David R. Liu. “A system for the continuous directed evolution of biomolecules” Nature 499, 2011.
Qiucen Zhang, Guillaume Lambert, David Liao, Hyunsung Kim, Kristelle Robin, Chih-kuan Tung, Nader Pourmand, Robert H. Austin. “Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments” Science 333, 2011.

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Tags: biocontrol, bioenergy, biofuel, biowaste, chromosome assembly, deep sequencing, food fungus, green energy, in vitro evolution, microenvironment, next generation sequencing, synthetic biology

Making a difference for those in need

This month 4 major pharmaceutical companies, GlaxoSmithKline, Merck, Johnson & Johnson and Sanofi-Aventis; all agreed to lower the cost of critical vaccines for developing countries. They have all done this as a part of the international vaccine alliance GAVI. The companies have severely slashed the prices of vaccines for diseases like rotavirus, a disease that isn’t prevalent in developed countries but causes more than half a million deaths per year. The model GAVI uses is one where vaccine costs are drastically lowered in developing countries, and this cost is offset by raised vaccine prices in developed countries.

GlaxoSmithKline has also reported that they are very close to developing an anti-malaria vaccine, which would be the first of its kind. This clearly shows a dissent from common pharmaceutical business practice, since Malaria is virtually wiped out in most developed countries. GSK has no hope of recouping costs for this vaccine by having patients in developed countries pay a premium for vaccination; but this has not deterred their efforts. Rather they have pledged to make on a 5% profit of the sale of the vaccine which will go toward future anti-malaria drug research.

Pharmaceutical companies are often viewed in a negative light for their practice of charging a premium for new drugs. However, the research, development, trials, and further clinical trials required to bring a drug or vaccine to market are all very costly, somewhat justifying a new drug’s high cost. Unfortunately this means there is no market for new drugs to combat diseases in developing countries as they cannot afford to compensate drug companies accordingly for their development costs. This is the key flaw in GAVI’s model, so it is great to see GSK is unhindered by this fact.

Everyday people who work in the biotech field strive to make a difference and help humanity through their research. Through the work of organizations like GAVI this research can ideally be utilized by all, and not just by those who can afford it.

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Tags: GlaxoSmithKline, healthcare, Johnson & Johnson, Merck, pour countries, Sanofi-Aventis, vaccines

Having trouble cloning?

Plasmid construction is constantly going on in nearly all molecular biology labs. Although nobody would like to describe cloning a piece of DNA into a vector as a major obstacle to a research plan in a grant application, or a glorious achievement in a publication; cloning could be, and often is the most time-consuming and mind-boggling step in a project. A typical theme in DNA construct creation starts with preparing a vector by restriction enzyme digest and insert DNA by either digest or PCR. The two pieces are then ligated together before transforming into competent bacterial cells where the ligated DNA molecules are amplified and selected.

The key to a successful execution of this procedure relies on retrieving correct DNA fragments before ligation. DNA isolation and recovery are currently done with PCR/gel extraction kit that utilize a silicon membrane immobilized inside a column, which can bind DNA (e.g. from a PCR reaction or a band cut out of a gel) in the presence of guanidinium. While this is a common practice in biological experiments, something often thought to be quite simple and straightforward; in reality it sometimes takes weeks or even months, repeat after repeat, before successful cloning is achieved. To increase cloning efficiency, people turn to “Super” competent cells, high concentration ligase, automated colony pickers, or high throughput sequencing for help.

Many sub-cloning projects get stuck due to plasmid recombination, by which a piece of DNA rearranges into a smaller plasmid than intended, often a bare-bone minimum plasmid that includes only the replication origin and antibiotic-resistance gene. This problem is amplified when either or both the vector and the insert fragments are large, or contain repeat sequences that destabilize DNA, such as those on viral vectors. Low efficiency of cloning is also a significant problem during library construction where a high degree of diversity is required. Recombination is facilitated by DNA nicks or breaks, something that can result from UV damage during gel viewing or by harsh chemical reagents in current DNA purification kits. The following is a recent real case of sub-cloning experienced by Allele Biotech researchers in our San Diego molecular biology lab:

Objective: cloning a group of 5 cDNAs (different versions or fragments from one gene transcript) into a retrovirus transfer plasmid for viral packaging
Vector: pCHAC1, ~12 kb, with terminal repeats
Insert: ~0.4-1.7 kb
Using standard PCR/gel purification kits (Allele Biotech), dozens or hundreds of colonies were obtained in each of the 5 rounds attempted, all of which were incorrect with various sizes below the projected size, including bare-bone (~3kb) plasmids. Different competent cells, (e.g. chemically competent DH5a, electro-competent DH10b), secondary structure-tolerant strains, etc. were tried to no avail.

Changes: Avoid all UV exposure and harsh chemical reagents, use solid surface binding that tethers DNA after each restriction digest or PCR directly in the coated PCR tube in the presence of a special binding buffer, and elute DNA into just the required volume of reaction buffer for the next reaction, e.g. ligation, transformation.

Results: 4 out of 5 constructs were made after only one round, with more than half of the colonies examined being correct. The failure of the 5th one was attributed to an orientation mistake in the parental plasmid used as PCR template.

Conclusion: DNA damage during gel running, cutting, and DNA extraction can severely hinder the creation of a difficult DNA construct.

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Tags: cloning difficulties, colony screening, competent cells, DNA cloning, DNA purification, DNA recombination, ligation, molecular cloning, plasmid construct, surface bind

Updated Publication List Using GFP-Trap Related Products

2011

Courtesey: list prepared by ChromoTek

Kastner, P. M., Schleicher, M., et al. (2011). The NDR Family Kinase NdrA of Dictyostelium Localizes to the Centrosome and Is Required for Efficient Phagocytosis. Traffic. 12: 301-312.

Guizetti, J., Schermelleh, L., et al. (2011). Cortical Constriction During Abscission Involves Helices of ESCRT-III-Dependent Filaments. Science.

Muhlen, S., Ruchaud-Sparagano, M. H., et al. (2011). Proteasome-independent Degradation of Canonical NF{kappa}B Complex Components by the NleC Protein of Pathogenic Escherichia coli. J Biol Chem. 286: 5100-5107.

Speck, J., Arndt, K. M., et al. (2011). Efficient phage display of intracellularly folded proteins mediated by the TAT pathway. Protein Eng Des Sel.

Heinrich, C., Gascon, S., et al. (2011). Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nat Protoc. 6: 214-228.

Qin, W., Leonhardt, H., et al. (2011). Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1. J Cell Biochem. 112: 439-444.

Shen, H., Ferguson, S. M., et al. (2011). Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin. Mol Biol Cell. 22: 493-502.

Wilkinson, K. A. and Henley, J. M. (2011). Analysis of metabotropic glutamate receptor 7 as a potential substrate for SUMOylation. Neurosci Lett.

Reininger, L., Wilkes, J. M., et al. (2011). An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony. Mol Microbiol. 79: 205-221.

Bubeck, D., Reijns, M. A., et al. (2011). PCNA directs type 2 RNase H activity on DNA replication and repair substrates. Nucleic Acids Res.

Dissanayake, K., Toth, R., et al. (2011). ERK/p90(RSK)/14-3-3 signalling has an impact on expression of PEA3 Ets transcription factors via the transcriptional repressor capicua. Biochem J. 433: 515-525.

Kuipers, M. A., Stasevich, T. J., et al. (2011). Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload. J Cell Biol. 192: 29-41.

Frauer, C., Rottach, A., et al. (2011). Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1. PLoS One. 6: e16627.

2010

Paris, L. L., Hu, J., et al. (2010). Regulation of Syk by phosphorylation on serine in the linker insert. J Biol Chem. 285: 39844-39854.

Korzeniowski, M. K., Manjarres, I. M., et al. (2010). Activation of STIM1-Orai1 involves an intramolecular switching mechanism. Sci Signal. 3: ra82.

Chamousset, D., De Wever, V., et al. (2010). RRP1B Targets PP1 to Mammalian Cell Nucleoli and is Associated with Pre-60S Ribosomal Subunits. Mol Biol Cell.

Thorslund, T., McIlwraith, M. J., et al. (2010). The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA. Nat Struct Mol Biol. 17: 1263-1265.

Erdel, F., Schubert, T., et al. (2010). Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites. Proc Natl Acad Sci U S A.

Geoffroy, M. C., Jaffray, E. G., et al. (2010). Arsenic-induced, SUMO-dependent Recruitment of RNF4 into PML Nuclear Bodies. Mol Biol Cell. (PudMed)

Boulon, S., Pradet-Balade, B., et al. (2010). HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol Cell. 39: 912-924.

Schmitz, K. M., Mayer, C., et al. (2010). Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev. 24: 2264-2269.

Bakondi, B. and Spees, J. L. (2010). Human CD133-derived bone marrow stromal cells establish ectopic hematopoietic microenvironments in immunodeficient mice. Biochem Biophys Res Commun. 400: 212-218.

Vermeulen, M., Eberl, H. C., et al. (2010). Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell. 142: 967-980.

Pozo-Guisado, E., Campbell, D. G., et al. (2010). Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry. J Cell Sci. 123: 3084-3093.

Kaidi, A., Weinert, B. T., et al. (2010). Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science. 329: 1348-1353.

Dzamko N., et al. (2010). Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J 430: 405-413.

Nichols R. J., et al. (2010). 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson’s disease-associated mutations and regulates cytoplasmic localization. Biochem J 430: 393-404.

Polo S. E., et al. (2010). Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4. EMBO J.

Babiano R., et al. (2010). Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 38: 5177-5192.

Loiseau P., et al. (2010). Drosophila PAT1 is required for Kinesin-1 to transport cargo and to maximize its motility. Development 137: 2763-2772.

Dubin, M., Fuchs, J., et al. (2010). Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis. Nucleic Acids Res.

Pabis, M., Neufeld, N., et al. (2010). Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20. Nucleus. 1: 412-421.

Van Dessel N., et al. (2010). The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets. Nucleic Acids Res.

Rass U., et al. (2010). Mechanism of Holliday junction resolution by the human GEN1 protein. Genes Dev 24: 1559-1569.

MacKay C., et al. (2010). Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142: 65-76.

Ommen G., et al. (2010). The co-chaperone SGT of Leishmania donovani is essential for the parasite’s viability. Cell Stress Chaperones 15: 443-455.

Fulcher A. J., et al. (2010). Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen. J Biol Chem 285: 17744-17753.

Taniue K., et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development 137: 1755-1764.

Kovanich, D., van der Heyden, M. A., et al. (2010). Sphingosine kinase interacting protein is an A-kinase anchoring protein specific for type I cAMP-dependent protein kinase. Chembiochem. 11: 963-971.

Boulon S., et al. (2010). Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics 9: 861-879.

Slabicki M., et al. (2010). A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol 8: e1000408.

Laxman, S., Sutter, B. M., et al. (2010). Behavior of a metabolic cycling population at the single cell level as visualized by fluorescent gene expression reporters. PLoS One. 5: e12595.

Bergbauer, M., Kalla, M., et al. (2010). CpG-methylation regulates a class of Epstein-Barr virus promoters. PLoS Pathog. 6.

Kalla M., et al. (2010). AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome. Proc Natl Acad Sci U S A 107: 850-855.

Bellanger S., et al. (2010). The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase. J Virol 84: 437-444.

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Recap from the San Diego Entrepreneur Exchange March Event on Green Energy

With the recent concerns about the safety of nuclear power originated from the Japan earthquake and Tsunami, it should be beneficial for us to recap what we have learned at the latest SDEE conference two days before the natural disaster hit Japan.

Ron Pitt, CEO of EcoDog:
Solar energy is a great source of energy but it’s limited by the supply of silicon. Furthermore solar panels have a life of about 20 years, at which point they need to be replaced. It’s important that we take steps to alleviate our dependence on oil and deal with the current crisis, but it is also imperative that we employ forward thinking, and expand the time scale so our fix doesn’t last 20 years, or even 200 years but much longer.

Barry Toyonaga, CBO of Kent BioEnergy:
Algea is probably the most efficient way of removing waste material in waters and to entice nutrition to soil. Even the biomass after use has been shown for making bricks in a recent conference in Japan. It is important to use every aspect of our raw material, we must be so efficient to the point where no useless waste is generated by the end of our process.

Steve Mayfield, Director of the San Diego Center for Algae Biotechnology, UCSD:
The energy generated from petroleum-derived fuels as well as chemicals are used for high efficiency production of food. The emission of CO2 peaked by many magnitudes in recent centuries and coincided with human population explosion. The fast depletion of oil will soon reduce humans’ abilities to produce food at such high efficiency, and unavoidably will lead to famine and population reduction. The recent unrest in Africa is not a fight for democracy but a fight for food (which we can’t agree).

The green industry is young and needs supporting roles even after high rollers like Sapphire Energy, a company spun out by Mayfield and recently received major equity investment from Monsanto, take all the spotlight. There are engineering work to be done to process the oil produced by algae, to manage production and transportation, etc.

Sandy Madigan, CEO of Strategic Enzyme Applications:
Lignin is a naturally present macromolecule in wood and other plants, it is very carbon rich and one of the few natural sources aromatic compounds can be derived from. If broken down effectively lignin can serve as an alternative source of carbon compounds, with the current source being petroleum. Furthermore, as a source of aromatics, it has the potential to provide an exact fuel replacement, as opposed to most current research looking for fuel alternatives.

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Tags: biofuel, green energy, Japan earthquake, melt down, nuclear power plant, renewable energy, tsunami, US West coast