The Strand - Synthetic Biology Newsletter, Issue 13
|What are our young scientists doing?
The Thermo Fisher Scientific–sponsored TP-CC (Torrey Pines High School and Canyon Crest Academy) San Diego iGEM team took part in this year's high school iGEM competition...read more
|GeneArt® PerfectMatch TAL
Precise and flexible genome editing...read more
|Tips & Tricks
Tips for designing CRISPR oligos for use with the GeneArt® CRISPR Nuclease Vector Kit...read more
Application of a synthetic combinatorial library for phage display...read more
CRISPR technology: top 10 questions
| Upcoming Events and Synthetic Biology Resources
Did you know?
LABTalks: our new video series
The first two videos of our new conference-style video series, LABTalks, are produced and ready for you to watch. This series of 15-minute videos has been created to give you a high-level introduction to our synthetic biology/genome engineering research tools, and is presented by one of Thermo Fisher Scientific’s experts in this area.
LABTalks: CRISPR/Cas9: Simple & versatile genome editing tool In this LABTalk Dr. Namritha Ravinder discusses the powerful new CRISPR/Cas9 technology for genome editing. It includes a general introduction to genome editing and applications as well as a specific introduction to CRISPR/Cas9 technology and an example of multiplexing the functionality of the CRISPR/Cas9 system.
LABTalks: Gene to protein; enhancing your protein yields Dr. Henry Chiou discusses combining the powerful technologies of gene optimization and transient expression for enhanced protein yields.
Learn about various parameters that influence your protein expression results
Hot from Social
The GENOME editors comic series
|What are our young scientists doing?
The Thermo Fisher Scientific–sponsored TP-CC (Torrey Pines High School and Canyon Crest Academy) San Diego iGEM team took part in this year's high school iGEM competition and finished in second place out of a total of 54 international high school iGEM teams. They also received the award for best poster.
What is iGEM?
The International Genetically Engineered Machine competition (iGEM) began in 2003 with a month-long course at MIT and has become the premier competition on synthetic biology for undergraduate and graduate students. The task for each team is to engineer living cells with novel and unusual properties by assembling existing or newly designed biological parts—so-called BioBricks. Since its founding iGEM has continued to grow, with 245 international teams in 2013. In 2011 iGEM expanded to include a high school (HS) division, with 5 teams in competition. HS iGEM expanded to include 30 teams in 2013 and 54 teams in 2014. There is no significant technical difference between HS iGEM and university iGEM.
What topic did the TP-CC San Diego team work on?
The aim of these young scientists was to produce mycotoxin-detoxifying enzymes (of fungal origin) in E. coli using synthetic biology and molecular biology tools (e.g., expression vector pTrcHis2_A, GeneArt® Seamless PLUS Cloning and Assembly Kits, and TOP10 and DH10B™ competent cells). Mycotoxins can cause severe health problems in humans—according to the WHO they are the third leading cause of cancer. In their experiments the students cloned and expressed two naturally occurring detoxifying enzymes (aflatoxin-detoxifizyme (ADTZ) and zearalenone hydrolase (ZHD101)) fused to secretion signal peptides in E. coli, with the long-term goal of generating high-yield recombinant forms—rapidly and cost-effectively.
Why is it important to sponsor the young scientists?
Thermo Fisher Scientist Chang-Ho Baek mentored the TP-CC San Diego team and says about the importance of sponsorship, “Actually the TP-CC San Diego iGEM team coordinator requested my mentorship, and I willingly accepted this offer, since I love to teach young scientists. When I visited a science laboratory at Torrey Pines High School, I realized that it’s a challenge for them to get the latest equipment and materials. In our first idea meeting for the team project this year, I could read their passion to learn about synthetic biology and to address important issues and problems on Earth such as pollution, diseases, and food. So these factors triggered my motivation to support them by mentoring and by arranging donation of materials from our company. I believe that sponsoring young scientists on the iGEM team will create an invaluable asset for society, because they have the potential to be the leaders needed to make our world cleaner, safer, and healthier in the future. Hopefully, our company will continue to sponsor iGEM teams with even greater generosity.”
Thermo Fisher Scientific also sponsors the university iGEM competition by the provision of discounted products for the teams.
If you’re interested in participating or in sponsorship, find out more about iGEM and the TP-CC San Diego iGEM team.
Send us your story on how we are enabling you to make breakthroughs.
|GeneArt® PerfectMatch TAL
Precise and flexible genome editing
Transcription activator-like (TAL) effectors are part of a widely used technology for precise and efficient gene editing in living cells. TAL effectors comprise a specific DNA-binding domain fused to an effector domain (nuclease, activator, or repressor), which allows specific gene modulation.
Up to now, the natural requirement of a 5’ T in the target sequence has limited the available targets. To overcome this limitation we mutated the N-terminal domain of our GeneArt® Precision TAL to create a T-less TAL. These new GeneArt® PerfectMatch TALs can be designed to bind to DNA sequences with any 5’ base (T, G, C, or A), anywhere in the genome.
The workflow for GeneArt® PerfectMatch TALs is similar to that of the current GeneArt® Precision TALs.
In order to characterize the functionality of GeneArt® PerfectMatch TALs, our R&D scientists have performed a series of experiments comparing the new GeneArt® PerfectMatch TALs fused to FokI nuclease with current GeneArt® Precision TALs, using different targets and cell lines (see examples below). The experimental outcome shows that GeneArt® PerfectMatch TALs exhibit activity equal to or better than our current GeneArt® Precision TALs. Detailed results can be found in the new Technical Product Guide, or on the GeneArt® PerfectMatch TAL web page (online soon).
The GeneArt® PerfectMatch TAL function is equal to or better than the current TAL effector in 293FT cells when targeting sequences of forward and reverse TAL effectors are preceded by different (non-identical) bases. The red arrows point to the cleavage products of the GCD enzyme if multiple bands were observed in a GCD assay. Note: GeneArt® PerfectMatch TALs functioned with at least 50% efficiency compared to GeneArt® Precision TALs, with the majority of PerfectMatch TALs showing better efficiency.
|Tips for designing CRISPR oligos for use with the GeneArt® CRISPR Nuclease Vector Kit|
To use the GeneArt® CRISPR Nuclease Vector Kit, first design two single-stranded DNA oligonucleotides (24–25 bp), one encoding the target-specific crRNA (forward-strand oligonucleotide) and the other its complement (reverse-strand oligonucleotide).
The choice of genomic target sequence can significantly affect the degree of cleavage observed. Make sure that the target sequence does not contain significant homology to other genes, as this can increase off-target effects. Recently published work has shown that gRNA-Cas9 complexes can potentially tolerate 1–3 or more mismatches, depending on their location in the gRNA (refer to the literature for more insights into choosing a target sequence). We recommend that you test more than one target-specific crRNA sequence per locus. Guidelines are provided below for choosing your target sequence, and an example is provided in the figure below. Note that these are general recommendations only, and you may need to make adjustments for your experiment.
Share your tips & tricks from your lab at SynBioNews@lifetech.com
Application of a synthetic combinatorial library for phage display
In this issue we would like to introduce, as an example of the use of a synthetic combinatorial library, the work of Dr. Andras Szabo et al., which was recently published in PLoS One. Dr. Szabo is at the Department of Molecular and Cell Biology at Boston University, Henry M. Goldman School of Dental Medicine, in Boston, Massachusetts, USA. His research goals include the characterization of human trypsins, which play a major role in digestion in all vertebrates.
Dr. Szabo, could you explain your research interest in a few words:
Our primary goal in the laboratory of Dr. Miklos Sahin-Toth is to elucidate the proteolytic regulation and interactions of pancreatic digestive enzymes and inhibitors that contribute to the pathogenesis of pancreatitis, an inflammatory disease of the pancreas.
In your recent publication you asked the question of the significance of trypsin sulfation at amino acid position 154 for the specificity of human trypsins. To address this question you first investigated the potential impact of sulfation on substrate recognition by trypsin from a structural perspective, and then decided to perform phage display experiments with a synthetic variant library of the bovine pancreatic trypsin inhibitor (BPTI), which binds in a substrate-like fashion.
Why did you decide to make a synthetic library?
By using a synthetic variant library based on the scaffold of BPTI, we were able to probe the specificity of sulfated trypsin at the positions that interact with the site of sulfation, in an unbiased fashion. The library was commissioned by our coauthor Dr. Evette Radisky for another project profiling the specificity of another protease in a study that is currently still ongoing. She decided on GeneArt® technology based on previous positive experiences with construction of other synthetic genes and libraries.
Within this library, which was produced at Life Technologies laboratories, five residues at different positions of BPTI were completely diversified and the library was cloned into a customer specific phage-display vector. The subsequent phage display experiments resulted in several tight-binding variants of BPTI, with all variations located at the same randomized amino acid position (P2’). These variants were isolated to investigate their specific binding affinity to sulfated and nonsulfated trypsin in more detail. Further analysis of the single mutants, by determination of the equilibrium dissociation constants, led to the conclusion that sulfation of human trypsin (at position 154) increases selectivity towards basic (Arg, Lys) versus apolar (Ala, Ile, Leu) residues at the P2’ position in inhibitors that bind in a substrate-like fashion. Although the biological significance of the observed effect of sulfation remains unclear, the authors speculate that sulfation of human trypsins at Tyr154 may have evolved to facilitate digestion of a broader range of dietary proteins.
Would you like to disclose your next steps to elucidate the biological function of sulfation?
Our future aim is to investigate the effects of trypsin sulfation on the solubility of the protein, and on its secretion by pancreatic acinar cells.
Thank you, Dr. Szabo, for sharing your thoughts, and all the best for your future research!
This interview was conducted by Nina Raab, Thermo Fisher Scientific Marketing Department.
Read the whole story: Tyrosine Sulfation of Human Trypsin Steers S2’ Subsite Selectivity Towards Basic Amino Acids
Learn more about GeneArt® Combinatorial Libraries
Recently published your data? Share your data with us and we’ll feature it in our newsletter.
Email us at SynBioNews@lifetech.com
|Key questions on gene optimization strategies for maximum protein expression|
Q. What is gene optimization, and how does it differ from codon optimization?
A. Our proprietary GeneOptimizer® software calculates the optimal DNA sequence needed to encode the protein of interest (gene optimization). Adapting the codon usage of the gene to the codon preferences of the expression organism (codon optimization) is just one of the parameters addressed. The GeneOptimizer® software currently evaluates up to 50 factors that can compromise mRNA stability, such as extreme GC content, ribosomal binding sites, consensus and cryptic splice sites, repeats, and secondary structures. An increase in the number of stable mRNA molecules often leads to a higher yield of protein.
Q. Do you consider cis-acting elements that increase protein expression when you optimize the codon?
A. We only optimize the open reading frames of your desired gene. We remove all regulatory elements wherever possible in order to avoid alternative transcription start sites. Negative cis-acting sites (e.g., splice sites, poly(A) signals, TATA boxes) which may negatively influence expression will be eliminated as well. We do not optimize promotors or other noncoding regions.
Q. What expression systems do you offer for codon optimization? Do you codon-optimize for expression in Schneider-2 Drosophila cells?
A. Yes, we routinely optimize according to the CUT (codon usage table) of Drosophila, which works well for Schneider-2 cells. We optimize to all CUTs available and to all standard expression hosts. If you would like to send us a special codon usage table for your cells, please contact the service team at GeneArtSupport@lifetech.com
Q. Can you optimize a genetic sequence for dual organism expression? For example, if I’d like to have optimized expression in either yeast or E. coli and want a sequence that will work well in both.
A. Dual optimization does not make sense in every case. It depends on the organisms you choose for expression, since their respective codon usages have to be compatible. Accordingly, dual optimization for E. coli and yeast expression would not be recommended because the most preferred codons in E. coli are more rarely used codons in yeast, and vice versa. The same would be true for E. coli or mammalian expression. On the other hand, more similar expression host pairs, such as Pichia and Saccharomyces, or human and hamster (CHO), will work very well for dual expression optimization. It clearly depends on each individual case, and we will advise you on the best solution for your project.
Q. What is the usual price range for the gene optimization service?
A. The gene optimization itself is free of charge. If you order a gene and use our online portal for optimization, you can decide if you want it synthesized as an optimized or wild type gene. We do not routinely offer gene optimization alone without Strings™ DNA fragments or gene synthesis.
Q. Can you enter your own codon usage table for gene optimization on the Life Technologies™ GeneArt® system?
A. Yes, you can submit your own codon usage table to us. Please contact GeneArtSupport@lifetech.com, and the support team will send you an Excel® spreadsheet where you can enter the desired CUT, and we will optimize your genes according to that CUT. It’s not possible to upload your own CUT to the online portal.
Q. Does gene expression optimization work with every protein, and can I also expect better yields if I already have good expression?
A. We know from our own data and from customer feedback that optimization can improve even proteins that are already expressed well. This is clearly different from protein to protein, but, for example, for antibody expression in 293 and CHO cells, we have seen large improvements, even if the antibody is already expressed well. However, if a protein does not express at all, we cannot guarantee that our optimization can change this, but it is definitely worth trying.
Read the results of our multigene expression study.
Q. Do you check for biosecurity?
A. Yes, we do a biosafety and biosecurity check for every gene we produce. That means we screen every sequence against a database for indexed sequences. If we find a hit, we may need clearance from local authorities to produce the gene. In such cases the customer will be informed that this clearance is needed and that it may be several weeks before the clearance is granted. In rare cases, we reserve the right to refuse the order due to a biosecurity issue.
Get more information about our gene optimization.
Watch our optimization videos:
Send us your questions at SynBioNews@lifetech.com