The Strand - Synthetic Biology Newsletter, Issue 10
Take a Shortcut to Your Gateway® Expression Vector
Life Technologies scientist Federico Katzen and his team published a cost- and time-saving method for cloning of your Gateway® expression vector.read more >>
Engineer your protein using directed evolution methods
Would you like to evolve your protein of interest but are tired of error-prone PCR approaches?
GeneArt® Genomic Cleavage Detection Kit
|An essential tool for monitoring the efficiency of your genome editing experiments|
Did you know?
$2.25 M in three years for six renowned universities, what’s that for?
It’s for the Semiconductor Synthetic Biology research program (SSB) launched and founded by the Semiconductor Research Corporation (SRC).
The aim of the program is to create hybrid bio-semiconductor systems by bringing electronics and biochemistry together with the help of synthetic biology approaches. In the long run, researchers hope that new cells can be invented, which can be integrated into hybrid biological semiconductors.
The program starts with three main areas of research including molecular-scale additive-chip fabrication processes, cytomorphic-semiconductor circuit design for new ultra-low-power microchip architectures, and invention of new bioelectric sensors, actuators, and energy sources.
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The Gateway® recombination system is a smart alternative to the traditional cloning method of restriction enzymes and ligase and is used to create a variety of expression vectors for your open reading frame.
Previously, the Gateway® protocol consisted of two separate cloning steps:
- BP reaction: A DNA fragment containing the gene of interest is recombined by BP Clonase® enzyme into a donor vector resulting in an entry clone.
- LR reaction: The entry clone is used to transfer the gene of interest into multiple destination vectors generating expression clones by use of LR Clonase® enzyme.
A number of applications do not benefit from an entry clone, the creation of which costs extra time and money. The authors developed a protocol which combines the two single recombination steps in one tube, resulting in the generation of both entry and expression clones. The experiment involves different ratios of BP and LR Clonase® enzymes followed by analysis of successful recombination events for the LacZα and GFP reporters.
The results show that single-tube BP and LR recombination is highly efficient and doesn’t require subsequent cloning steps. If the exclusive goal is to obtain an expression clone, the use of LR Clonase® enzyme alone is preferred as it can perform both recombination events. There are still a reduced number of entry clones available. Furthermore, this approach can be used for the generation of an expression vector containing multiple fragments or for transfer of an insert from one expression clone to a second destination vector in one step. Taken together, performing one reaction typically reduces your cloning time from three days to just a single day, and the price per reaction is reduced because you use less BP Clonase® enzyme.
The editors of BioTechniques cited our publication as one of their three favorite PCR/Cloning papers from 2013.
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Tips & Tricks
Engineer your protein using directed evolution methodsWould you like to evolve your protein of interest but are tired of error-prone PCR approaches? Check out the GeneArt® Directed Evolution methods for protein engineering webinar and learn time-, effort-, and cost- effective alternatives for your protein research. This webinar includes an exemplary case study comparing traditional error-prone PCR method versus a rationally designed library.
Synthetic biology has now paved the way to create genes and libraries that require no natural template at all—almost any design vision is possible. Synthetic genes are well suited for optimizing protein expression in a non-natural host or to acquire genes that are hard to find in natural sources. DNA synthesis is based on the orderly assembly of short, chemically produced oligonucleotides. The same methodology can also be used to synthesize a library that mimics the error-prone PCR. During automated oligonucleotide synthesis, a definable frequency of misincorporation of an alternative residue is allowed for certain positions by adding distinct impurities to the corresponding reagent bottle. Based on these ‘spiked’ oligonucleotides, a controlled randomization library can be assembled:
- That has the statistically expected ratio of transitions and transversions
- Where the average number of substitutions can be fine-tuned very accurately
- Where the positions of possible substitutions are not entirely random anymore but limited to regions of interest defined solely by the investigator
How many mutations should one variant carry on average?
In general, a good rule of thumb is to limit the complexity of the library to still allow for the occurrence of the wild type gene (without mutations) in the screened subset. This helps ensure that the examined variants don’t diverge too much from the original protein, which could result in a loss of function for the complete pool.
Definition of regions of interest for which mutations should be introduced
This question can be approached in many different ways, depending on the individual project. If the three dimensional structure of the investigated protein (or a highly similar one) is available, the amino acid residues likely related to the protein’s function (for example the active site of an enzyme) can sometimes be deduced and confirmed by single point mutations. If this data is available, it is a good idea to limit randomness to only these positions and to those that are in close three-dimensional proximity in a degenerated library. The remaining scaffold of the protein may be left unmodified.
Another approach uses the availability of natural homologous sequences in protein or DNA databases. These can be aligned according to their similarity to identify regions of conservation and variation, reflecting the natural selection pressure for the particular gene family. Depending on the overall similarity within the family and the scope of the aimed protein adaptation, a library design will provide for new mutations either in the conserved or the variable part of the gene. One example is antibodies which have a very conserved framework, but are highly variable within the antigen-binding pockets and are best randomized in these regions to identify new binding specificities.
If no such data is available, or if it does not correlate with the protein performance, the protein function topology can be mapped ahead of designing evolution experiments. A common tool is an alanine scan which comprises the generation of all consecutive variants with each single amino acid being changed to alanine. The testing of these variants for maintenance or loss of function is an indicator for the importance of each protein position and usually identifies clusters of interest for further mutagenesis. The extreme application of this strategy is to change each individual amino acid not only to alanine but to all 19 non–wild type residues. For this 'sequential permutation' of a 300 amino acid protein, it involves the screening of 300 x 19 = 5,700 variants, which is manageable, even with low-throughput assays.
The functional analysis of these mutants generates a data matrix containing information about the importance of each amino acid position regarding overall function, and which non–wild type amino acid contributes to the adaptation of the protein towards the technical demands. In this type of experiment it is also common to identify beneficial mutations at amino acid positions that were not previously considered to be related to protein performance. With this valuable information at hand, it is possible to combine the advancing single mutations in a new combinatorial library to screen for synergistic beneficial effects. These second-round libraries typically concentrate on not more than 10 sites with either the wild type or the improving amino acid present. Thus, the size of this library is only ~210 = 1024, and can be entirely screened even in complicated assays. As the demand for adapted proteins for industrial and medical applications increases, so will the scope of approaches to tailor them.
Visit our Directed Evolution website >>
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GeneArt® Genomic Cleavage Detection Kit
An essential tool for monitoring the efficiency of your genome editing experiments
When using genome editing tools such as CRISPRs, TALs, or zinc finger nucleases to obtain targeted mutations, it is necessary to determine the efficiency with which these nucleases cleave the target sequence prior to continuing with labor-intensive and expensive experiments. The GeneArt® Genomic Cleavage Detection Kit provides a simple, reliable, and rapid method to determine the nuclease cleavage efficiency at a given locus.
The GeneArt® Genomic Cleavage Detection Kit contains most of the reagents you’ll need, except for primers and those needed for gel analysis. A sample of the edited cell population is used as a direct PCR template for amplification with primers specific to the targeted region. The PCR product is then denatured and reannealed to produce heteroduplex mismatches where double strand breaks have occurred, resulting in indel introduction. These mismatches are recognized and cleaved by the detection enzyme. This cleavage is both easily detectable and quantifiable using gel analysis (Figure 1).
Figure 1. Gel images of cleavage assays performed with the GeneArt® Genomic Cleavage Detection Kit (Cat. No. A24372). (A) Results using GeneArt® Precision TALs targeting the AAVS locus. (B) Results using the GeneArt® CRISPR Nuclease CD4 Vector targeting the HPRT locus. Following transfection into HeLa cells, triplicate cleavage assays were performed and the percentage of indels was calculated.
Release of siRNA library sequences enable scientists to identify new potential drug targets
One efficient approach to investigate regulators of cellular processes is to perform a genome-wide RNAi screen to identify possible players involved in certain pathways or interactions. A limiting factor of this approach is the availability of proprietary siRNA library sequence information by providers. A solution is now offered by NCATS (the National Center for Advancing Translational Sciences at the NIH) and Life Technologies, establishing the public-private collaboration called the NIH RNAi initiative. Life Technologies provides siRNA sequence data from its Silencer® Select siRNA library, which includes 65,000 siRNA sequences targeting more than 20,000 human genes; NCATS publishes data on the effects of the siRNAs on biological function in a free accessible database.
An exciting exemplary article using this siRNA library information was published last November by Hasson et al., searching for regulators of Parkinson’s disease–associated proteins PINK1 and Parkin. In their natural environment these proteins help prevent accumulation of dysfunctional mitochondria. To identify new regulators of PINK1-dependent Parkin translocation to damaged mitochondria, the scientists used two different siRNA libraries. The automated screen yielded a long list of possible interacting candidates that were further assayed and analyzed for off-target effects. Finally four candidates—TOMM7, HSPA1L, BAG4, SIAH3—were subjected to deeper investigation.
To exclude off-target effects the authors applied several approaches to specifically knock down the respective genes including TALEN-mediated genome editing and lentiviral shRNAs. Further experiments on the four genes made these hot candidates for new targets for treating Parkinson’s disease or other neurological disorders.
Overall, using siRNA library screens with available sequence information allows researchers to identify subsets of potential drug target candidates. Furthermore the NIH invites all companies selling siRNAs or scientists working with the Life Technologies siRNA library to deposit sequence data and biological activity information into the NIH database.
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Questions & Answers
GeneArt® Strings™ DNA Fragments frequently asked questions
Q. What are Strings™ DNA Fragments?
A. GeneArt® Strings™ DNA Fragments are custom-made linear, double-stranded DNA fragments up to 3,000 bp in length, ready for cloning in your lab. Strings™ DNA Fragments are assembled from synthetic oligonucleotides using the same process developed for GeneArt® high-quality gene synthesis. At least 200 ng of Strings™ DNA Fragments, up to 1 kb, are typically shipped within 5 business days, or 8 business days for 1–3 kb. GeneArt® Strings™ DNA Fragments can be ordered through our electronic web order portal and offer a fast and convenient alternative to full gene synthesis that is affordable for every lab. Try our new GeneArt® Type IIs Assembly Kits for cloning your Strings™ Fragments!
Q. How are Strings™ DNA Fragments quality-controlled?
A. Strings™ Fragments are PCR amplicons of assembled oligonucleotides, and an intermediate product of the gene synthesis process. Thus, the DNA fragments in the pool are not identical, and Strings™ Fragments are not 100% correct like a cloned gene synthesis product. Strings™ DNA Fragments are bulk sequenced to help ensure that your desired sequence is highly represented in the pool and you can find a correct clone by screening.
Q. Are there any gene editing and gene optimization functions available when ordering Strings™ DNA Fragments?
A. Yes. We recommend using the electronic web order portal for ordering, where you can use the free, convenient gene editing and optimization functions to adjust the sequence according to your exact needs (e.g., to add the desired end sequences for subcloning, or suitable sequences for assembly of gene fragments into a larger gene sequence).
After editing and optimization, please check that the final sequences of the fragment(s) are exactly as needed for further processing in your lab (e.g., potential homologous overlaps of the fragments or restriction sites and buffer bases).
Q. Do you offer services for subcloning or assembly of Strings™ DNA Fragments into larger genes?
A. No, subcloning and assembly services are not available for Strings™ DNA Fragments, as they are designed to offer you the fastest and most affordable way to get access to your genes. If you do not want to clone your gene yourself, we recommend that you order gene synthesis.
Q. How are Strings™ DNA Fragments delivered?
A. Strings™ DNA Fragments are delivered dried, ready for resuspension and cloning. Freshly resuspended Strings™ DNA Fragments should be used immediately. For longer storage, Strings™ DNA Fragments should be dispensed into aliquots and frozen at –20°C.
Q. Is the delivery time of all length categories of Strings™ DNA Fragments the same?
A. No. Strings™ DNA Fragments up to 1 kb are shipped typically within 5 business days, whereas Strings™ DNA Fragments between 1–3 kb are shipped within 8 business days. Delivery time varies depending on location.
Q. Can I order all my genes as Strings™ DNA Fragments instead of ordering gene synthesis?
A. No. Strings™ DNA Fragments are uncloned and limited to a length of 3,000 bp. If your gene is longer, you will have to assemble it from two or more Strings™ DNA Fragments.
In addition, some sequences may be too complex to be produced as Strings™ DNA Fragments due to repetitions or high GC content. In that case, you’ll receive a message and recommendation on how to order your gene via gene synthesis.
Q. Are Strings™ DNA Fragments always cheaper than gene synthesis?
A. Yes. Since you need to perform the cloning to obtain your final gene, Strings™ DNA Fragments prices are always lower than those for gene synthesis.
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