Stay in the know about gene synthesis services—see the complete webinar Q&A
Your colleagues submitted interesting questions during the webinar, “Move beyond traditional DNA cloning and editing,” so we’ve collected and answered them here.
Find the answer quickly by clicking your desired question.
Q: What are major limitations on size that can be synthesized and how will that be change over next few years?
A: Today, a 10kb gene or longer is built as part of the regular production process whilst its synthesis had been really challenging ten years ago. Standard processes cover up to 60kb now and what we can do easily in our R&D labs is 100kb. Indeed, the major limitations on size come from E. coli as the DNA propagation host. When the size gets significant compared to it's own genome, E. coli gets into problems. This can be overcome by switching to other suited hosts, e. g. like yeast. We expect size limitations to further diminish in the future and expect megabase sizes to be more or less easy to get synthesized within the upcoming years.
Q: Do you have the price chart and delivery times for the DNA strings? Maybe I missed it.
A: Check here for pricing of GeneArt® Strings™ DNA Fragments.
Q: How successful are you at synthesizing genes with really high G/C content?
A: We have an extremely low failure rate, which is about one out of five thousand or ten thousand genes, most probably due to toxicity of a gene and not because of high GC content. High GC constructs indeed are more demanding but nevertheless part of the routine production, based on a special technique, which takes one week longer but is very reliable.
Q: Do you have a minimal size "gene" you can make?
A: We have a very short minimum length defined if it is about our physical processes but this does not matter for your orders. By building a short DNA with flanking regions mimicking a vector's backbone we can represent a theoretical insert of only one base pair in length.
Q: Can I use Lasergene to order (we don't have access to Vector NTI®)?
A: An ordering interface is only for Vector NTI®, however you may download a free trial, click here or you may try to copy and paste the sequence if working with Lasergene.
Q: Can sequences be too complex to be made as GeneArt® Strings™ DNA Fragments? Then what?
A: Yes, this can happen. A low number of sequences will be too hard to be synthesized as GeneArt® Strings™ DNA Fragments. Since this evaluation happens before you can order, you will receive a message and recommendation on how to order your gene via gene synthesis instead.
Q: What is the longest turnaround time that is usual?
A: Turnaround time basically depends on the length of a gene and its complexity. The majority of genes are between 1 kb and 3 kb, which is the size of a regular human ORF. Standard turnaround time here is 10 to 15 business days. If an ORF of that length encodes complex and demanding sequence parts it might take about one week longer to get it finalized.
Q: Will a recording of this webinar be available later?
A: Yes. Please view this webinar along with the entire Synthetic Biology webinar series at www.lifetechnologies.com/synbiowebinars.
Q: Is 10 days realistic for a complex plasmid with, say a custom backbone?
A: If the gene qualifies to get done as SuperSPEED, it can be produced in five business days and then we do the subcloning in additional five days. So under certain favorable conditions there is a chance to get it done in 10 days despite of the complexity. In case of unfavorable conditions it might take 20 business days.
Q: Can you guys perform a small-scale expression to prove that the optimized gene is actually better than the WT submitted to you? What is the proof that I am getting value for my money?
A: We could offer expression tests based on a fee for service model. However because of the nice expression results of our optimized genes there never was a requirement for such an expression proof. We have literally thousands of customers including probably all of the biggest Pharmaceutical companies, and none of them have asked us to redo an optimization yet.
Q: I want to do transfection assays. Can you deliver larger amounts and the required quality?
A: Yes, we can deliver that. We offer endotoxin free plasmid preparation that can be directly used for transfection experiments. One of our most popular solutions here is our "Ready-to-work" package which includes cloning into your expression vector of choice plus delivery of one 1 mg endotoxin free DNA on top of your gene synthesis.
Q: Can you give us more information about what is meant by flexibility in design if talking about gene synthesis?
A: We are talking about flexibility when you are actually in the power of shaping your gene to exactly your purpose.
Lets consider a real example where a researcher failed to clone an 800 bp fragment into a fairly large vector. The non-directional, blunt end cloning strategy that had been chosen did not succeed through months. Actually with de novo synthesis the researcher was able to remove the blunt end sites and add well-performing standard cloning sites instead for directional cloning. Synthesis and subcloning went well in our hands and were done in the very short time period of three weeks despite the large vector backbone.
The gene synthesis approach could have saved enormous amounts of time and energy if chosen as strategy from the beginning.
Q: We saw a nice example of mammalian optimization. How about E.coli?
A: We also conducted a large study using E.coli genes, optimized for E.coli. This was published in 2010 with very similar results. For more information, see here http://www.ncbi.nlm.nih.gov/pubmed/20506237