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.

New Products of the week: Modified UTP (Pseudouridine-5´-triphosphate), and Modified CTP (Methylcytidine-5´-triphosphate) for in vitro transcription of mRNA.

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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

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

Making primers

We often hear that oligos (primers) are a simple product, and they should work every single time. From a producer’s point of view, oligos are arguably the most complicated products among common molecular biology reagents, at least in terms of the number of chemical steps required. DNA synthesis starts from the 3’ end to the 5’ end (opposite to DNA polymerases) on a solid support (e.g. CPG beads). For the addition of each base, the process begins by removing a protection group via “Deblocking”; then activating the last base for coupling with an “Activator”, adding the current nucleotide in the chemical form of a phosphoramidite (4 times in our protocol), blocking un-reacted openings with “Capping A and B solutions” (again 4 times each), forming bonds between bases by oxidization with Iodine, then looping all the way back to the beginning of the cycle. Many of these chemicals are either highly sensitive to moisture or have a short shelf life (can go bad any time).

Coming back from a seminar, sitting at the lab desk, you know you have a new idea and some cloning to do, and of course, it must be done tomorrow. The first thing you do is to send in some oligo sequences online to a local synthesis company late in the afternoon after looking at some maps and sequences. Around noon the next day, the oligos will be delivered in person to your hands. Most times everything will just work out fine as far as experiments involving primers are concerned; others you get stuck here and there along the way of cloning. Chances are you have run into problems with primers not giving PCR signals or clones with mistakes in the primer regions at some points in your research career. Even if this has only happened a few times, the memory, as well as the dissatisfaction and anger, can last for quite some time.

Between ordering and delivering, oligos are made overnight; they are then post-synthesis processed (requiring several hours starting in the early morning), OD’ed, and concentration adjusted. Given that the machine completes the run without any problems (power or computer related), and none of the chemicals run out, the best quick indicator of a good run is a color change from the protection group removed by Deblock at the last base. If there is visible amount of blocking group at the end of the synthesis, as reflected by an orange color from a Trityl group, it is likely that the synthesis was efficient till the end. Unfortunately, the efficiency of adding each base is never 100%–accumulations of missing or, at a lower percentage, mistaken bases will add up, especially in long oligos. Purification will remove some of the oligos with deletions, but not all of the bad oligos. MASS analysis will help determine the approximate percentage of bad oligos, but it will require time and cost not typically chosen by customers. It is our hope that understanding how oligos are made will help with more effective use of oligos when you order oligos, conduct experiments using oligos, or clone with oligos.

New Product of the Week: Phosphate-3′-CPG for oligo synthesis, email oligo@allelebiotech.com for details.

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Tags: amidite, amino oligos, biotin oligos, CPGs, FAM amidite, fast turnaround, insert, oligo, oligo chemicals suppliers, PAGE, PCR, primer, probe, siRNA, trityl

Purifying DNA without Membrane Binding

Purifying DNA from natural samples or biochemical reactions is one the most frequently performed experiments in virtually all molecular biology labs. Binding DNA to silica membranes in chaotropic binding buffers is the currently prevailing method, pioneered by Qiagen. Before Qiagen columns and the similar columns from a number of companies, including Allele, there was the silica resin, mostly from Promega. Before silica, it was phenol extraction or CsCl gradient.

Silica-based technology has been around for more than a decade, and it is time for a new generation of technologies that are more convenient and efficient than silica membrane to take center stage of DNA purification, especially given the fast-paced advances in polynucleotide analysis in microarrays and deep sequencing. Solid Surface Reversible Binding (SSRB) technology should be a shining star in coming years. The process is simple: DNA or RNA molecules in a simple binding buffer bind to the surface of plastics of any size and shape (PCR tube, 2.0ml eppendorf, 96-well plates, even 15 ml or 50 ml conical tubes) that is treated by a special process, washed, and eluted in any volume of water or even downstream reaction buffers. The utmost convenience is that the downstream reaction can be performed in the same tube!

This process is different from the electroreversion type of binding and releasing that requires buffers of different and extreme pH. Allele Biotech has started marketing SurfaceBind PCR purification kits, and will roll out products that are specifically tailored for genomic DNA, mRNA, size-differentiated DNA or RNA, DNA or RNA from fixed samples, from different species, etc. The convenience and cost-efficiency of these systems will provide significant contributions to the broad scientific community.

New Product of the Week 101810-102410:

SurfaceBind PCR purification kit, questions? Please email us at oligo@allelebiotech.com for a product description and introduction quotes.

Promotion of the Week 101810-102410:

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Tags: deep sequencing, DNA purification, genomic DNA, microarrey DNA sample, miniprep, miRNA, nextgen sequencing, PCR cleanup, PFA linked, RNA purification, silica membrane, surface resersible binding

3 Ways of Making DNA Libraries through Oligo Synthesis

Pools of DNA molecules of related but non-identical sequences are often used for selecting cDNAs that encode polypeptides with desired functions (such as in antibody screening), or DNA segments as protein binding sites (through SELEX), or DNA molecules that can catalyze reactions (DNA enzymes or deoxyribozymes), etc. The most direct way of creating DNA libraries is to introduce mixed bases during the synthesis of the oligos that will be used in creating the libraries.

1) The most commonly used method of generating degenerate oligos is to use mixed phosphoramidites (aka amidites, the building blocks of oligo synthesis) at desired positions in an oligo, e.g. using “N” to incorporate dA, dC, dG, and dT nucleotides, or “Y” for pyrimidines, “R” for purines. Mixed base oligos from most oligo suppliers are simple to order (and at no extra charge from Allele and a few other sources). During automated chemical synthesis of oligos, the synthesizer consecutively adds dT, dA, dC, or dG in the case of “N” at a pre-set ratio (e.g.25% each). This procedure does not always result in expected usage of each amidite because different amidites have different coupling efficiency, and the order of addition may also bias against amidites that are added later.

2) Using mixed bases like in method 1) leaves little control to achieve ratios of codons for specific amino acids. On the other hand, by using trimer amidites, which can be used for adding 3 nucleotides in each synthesis cycle, one can create oligos encoding selected amino acids at pre-determined percentages. However, this procedure is difficult to perform because trimer amidites are bulky and hard to couple to the elongating oligo; any moisture present during synthesis would have even more severe adverse effects than with regular amidites. Trimer oligo synthesis projects cost several thousand dollars per oligo on materials alone, and the risk is quite high that the oligos would not turn out of desired properties and qualities. For commercial users, this process has another problem—it is patented.

3) Another method for making library oligos is the so called “split-and-pool”, which is particularly suitable for having diversified amino acids embedded in otherwise common sequences like the CDRs within antibody variable regions. The latest oligo we made last month was a ~72 nt oligo with 8 locations that have pre-determined composition of amino acids, i.e. 20% Ala, 10% Gly, 12% His, etc. The procedure took us about 8 hours and we estimated the cost to be about $1,000. The subsequent sequencing results confirmed that ~70% of the clones using this oligo have desired degeneracy, compared to a similar oligo made by a bigger oligo company, at only 40%. In addition, we did not see any stop codon interruptions or major abnormalities.

DNA pools can also be generated by error-prone PCR, or more specifically with overlapping PCR using degenerate primers. The bottleneck for a library screening is how to handle big enough a number of colonies to accommodate the population, e.g. 10e10, or at least 10e8 clones are needed for finding high affinity antibodies. The second critical point is to have a robust and consistent selection readout such as fluorescence in cell sorting.

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Tags: antibody, CDR, degenerate oligos, DNA library, DNA primer, fluorescence, mixed bases, oligo, oligos, split-and-pool, trimer oligo