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You and your colleagues submitted interesting questions during the webinar, “Directed Evolution Methods for Protein Engineering,” so we’ve collected and answered them here.

 
Find the answer quickly by clicking your desired question.

Q- What will be the next-generation strategy?

Q- How much would it cost to saturate all positions of an average 300 amino acid protein?

Q- What is the typical turnaround time from ordering to receiving the gene?

Q- What does X represent here in TRIM

Q- Is there a diversity bias during cloning in E. coli?

Q- Dr Liss, what type of mutations would you recommend when trying to improve the thermostability of an enzyme while preserving its activity.

Q- With this method (screening for single site changes then combinatorial ones), how likely is it that you will miss a combination of mutations that produce a synergistic effect, but not individual effects for either change alone?

Q- Can we have access to the PowerPoint™ slides after the webinar?

Q- In the rational GFP approach, an SHG codon was a blue FP. Did these amino acids already exist in the known GFPs, or was it a new amino acid sequence? Or, would you have found it when you would have used only a rational approach without randomizing?

Q- What did you screen for, improved fluorescence in whole cells?

Q- What is the minimum percent of a particular amino acid that can be specified for a combinatiorial library? And how accurate is this?

Q- Again concerning the pricing, what would the GFP project cost for a customer?

Q- Can you answer what the cost is per site for site-saturation mutagenesis? I can't find this on the Life Tech website.

Q- How do we download this webinar to replay in my lab?

Q- Can you clone the library into any vector, or do you have your specialized vectors?

Q- Do you promise the same transformation rate (10^9) for bacillus as for coli?

Q- Can you please repeat the reason as to why NNS is prone to out-of-frame mutations?

Q- Is there literature available on improving thermostability of a protein published by GeneArt®, or tutorials on the Life Technologies websites?

Q- What are the limitations in terms of number of continuous positions (e.g., in an antibody CDR) that can be randomized by TRIM?

Q- Cost and turnaround?

Q- Are the 3' and 5' bases of the fragment ever variable in sequence (even when they are designed to be constant)?

Q- Can you please give a rough estimate of the cost for site saturation mutagenesis; the per site cost?

Q- Is your pricing model a fee for service, or is some licensing required? Is your combinatorial approach free of intellectual property?

Q- What aspects of the library design contribute the most to lower library correctness? (such that such design features could be avoided to allow a better quality library)?

Q- How about an actual estimate of the price? $500, $5000, $50000 per site?

Q- What type of structural data is available? Is this only with comparison to current knowledge, or can one compare changes in aa residues to changes in structure?

Q- I'm confused- is the TRIM technology any different than having oligos constructed with the mixtures of trinucleotides? We have had this done with synthetic codons from Glen research.

Q- Is it possible to control single amino acid mutations in order to study 'KO' mutations of the protein?

Q- The customization is combinatorial? I lost connection and am not sure if I missed how you are able to customize specific amino acid at a specific position?

Q- Are libraries available in Gateway® expression constructs?

Q- Do you offer codon optimization? If so, how? Are you good at it? References?

Q- Can different length mutagenic regions be incorporated together into a single library of fragments (for a single order?)

Q- What organisms are you able to transfect into for specialized customer vectors? Is it only E. coli and B. subtilis?

Q- Are you able to ensure that no wild-type proteins sequence remains after mutagenesis?

Q- Can a screen of a library following the mutation be done for a decrease in protein activity? If one is looking for a null mutant versus an improvement?

 

 

Q-What will be the next-generation strategy?

A- Next-generation strategies will probably involve the de novo synthesis of constructs within the library by massive parallel construct production using chip based technologies. Chip based screening methods might also play a vital role in the future.

 

Q- How much would it cost to saturate all positions of an average 300 amino acid protein?

A- It depends on the deliverables you choose. If you are choosing a pooled delivery where you find all single substitution variants in one tube, the costs will be around 30,000 USD. For an average of 16 mutations per site with single construct delivery, the cost will be 8 to 10 times higher.

 

Q- What is the typical turnaround time from ordering to receiving the gene?

A- Typical turnaround times for non-cloned libraries are 4- 6 weeks, while the delivery of a cloned library might take up to 8 weeks.

 

 

Q- What does X represent here in TRIM?

A- X should simply show that there is a mixture of codons used to generate this position.


 

Q- Is there a diversity bias during cloning in E. coli?

A-During all production steps we avoid growing E. coli in liquid culture. Instead, all growth of biomass happens solely on plates, which diminishes competitive growth and greatly increases equal distribution of the specimen in plasmid preparation and glycerol stocks. We can not avoid or rule out under representation of specimens that have a toxic effect in E. coli.


 

Q- Dr Liss, what type of mutations would you recommend when trying to improve the thermostability of an enzyme while preserving its activity.

A-It is always hard to predict which mutations are going to do what. Therefore the site-saturation mutagenesis (SSM) solution is ideal to test all single substitutions for their respective influence on a certain protein property. From our customer example on SSM, you can see that beneficial substitutions even at a designated protein position can come from substitutions belonging to different chemical groups (polar, uncharged etc.). So it is very hard to predict.

 

 

Q- With this method (screening for single site changes then combinatorial ones), how likely is it that you will miss a combination of mutations that produce a synergistic effect, but not individual effects for either change alone ?

A- It is very unlikely that you will miss a synergistic effect of mutations that (i) correspond to the sites you already identified in the first round and are (ii) are either wildtype or one of the beneficial mutations. All these combinations will be present in the screen. However, it is likely that you miss a synergistic effect of a neutral mutation in combination with another neutral or even another beneficial mutation. Neutral mutations will simply not be included in the second-phase combinatorial library. There is currently no reliable method in place that solves this problem effectively.

 

Q- Can we have access to the PowerPoint™ slides after the webinar?

A- The webinar can be revisited on demand here.

 

Q- In the rational GFP approach, an SHG codon was a blue FP. Did these amino acids already exist in the known GFPs, or was it a new amino acid sequence? Or, would you have found it when you would have used only a rational approach without randomizing?

A- " SHG is an already known motif, representing the fluorophore in BFP. We have taken these amino acids into account in creating the rationally designed library. Therefore, we have not selected a novel, unexpected phenotype and/or aa motif but an anticipated one. A proof-of-concept, if you wish."

 

Q- What did you screen for, improved fluorescence in whole cells?

A- Yes, we did screen for fluorescence in whole cells. A reason for the stronger fluorescent phenotypes might also have been stronger expression due to the introduced mutations.

 

Q- What is the minimum percent of a particular amino acid that can be specified for a combinatorial library? And how accurate is this?

A- The minimum reasonable amount is around 1%. If you use the chi-squared test, it usually fits pretty good for most of the codons.

 

Q- Again concerning the pricing, what would the GFP project cost for a customer?

A- The library for the GFP project would have probably cost around 10,000.00 USD.

 

Q- Can you answer what the cost is per site for site-saturation mutagenesis? I can't find this on the Life Technologies website.

A- It depends on the deliverables you choose. If you are choosing a pooled delivery where you find all single substitution variants in one tube the costs for a 300 amino acid protein will be around 30,000.00 USD. For an average of 16 mutations per site with single construct delivery the cost will be 8 to 10 times higher.


Q- How do we download this webinar to replay in my lab?

A-The webinar can be revisited as webinar on demand here.

 

 

Q- Can you clone the library into any vector, or do you have your specialized vectors?

A- Libraries can be cloned in any type of vector as long as it is suitable for high-throughput cloning. Most of the libraries are actually cloned into customer vectors.

 

Q- Do you promise the same transformation rate (10^9) for bacillus as for coli?

A- The transformation rates of 10^9 are usually only achievable with highly electrocompetent E. coli strains. We could also transform into bacillus. However, this can only be done into cells the customer initially delivers. Moreover, transformation rates will be significantly lower.

 

Q-Can you please repeat the reason as to why NNS is prone to out-of-frame mutations?

A- It is not the NNS itself that makes it prone to out of frame mutations. However, the most common error during primer synthesis is deletions. During regular primer synthesis bases are added one by one. Deletions are created very seldom, but if you face a deletion it leads to an out-of-frame mutation. During TRIM synthesis you add triplets. If a triplet does not get incorporated, a deletion will be created. The deletion will be in-frame since the whole codon gets lost.

 

Q- Is there literature available on improving thermostability of a protein published by GeneArt®, or tutorials on the Life Technologies websites?

A- Unfortunately we do not have any customer examples on thermostability improvements. However, we know that several customers did that successfully.


 

Q- What are the limitations in terms of number of continuous positions (e.g., in an antibody CDR) that can be randomized by TRIM?

A- The technical limitations are 15-20 adjacent codons randomized with TRIM. However, the quality is slightly worse for regions of that length (stretch of 20 randomized triplets).


 

Q-Cost and turnaround?

A- The pricing for combinatorial library libraries is very project dependent. Different factors like cloning (yes/or no) number of randomized codons and the type of randomization do influence the pricing. Please send us a complete sequence and we can quickly provide you with a cost estimate. Typical turnaround times for non cloned libraries are 4- 6 weeks while the delivery of a cloned library might take up to 8 weeks.


 

Q- Are the 3' and 5' bases of the fragment ever variable in sequence (even when they are designed to be constant)?

A- The error rate in the constant regions of a library is very low. We are aiming at an overall correctness of a library that is higher then 80%. Most of the occurring undesired mutations exist within the randomized region. The constant parts might carry 1-3 % of the overall occurring unwanted mutations.


 

Q- Can you please give a rough estimate of the cost for site saturation mutagenesis; the per site cost?

A- It depends on the deliverables you are choosing and how many sites you want to address. Let me give you 2 examples. If you would like to receive all single substitution variants in one tube pricing ranges from 100-150 USD per site. If you request the delivery of an average of 16 mutations per site delivered as single constructs on 96 well plates costs will be between 800-1,200 USD per site.


 

Q- Is your pricing model a fee for service, or is some licensing required? Is your combinatorial approach free of intellectual property?

A- In general we do not require licensing. For details please review the gene synthesis terms and conditions which will be provided with a quote. If you like to receive those before hand please let me know.


 

Q- What aspects of the library design contribute the most to lower library correctness? (such that such design features could be avoided to allow a better quality library)?

A- As a general rule of thumb you can say that the more random codons that are directly adjacent the lower the quality of the library. Especially stretches with more than 15 directly adjacent randomized codons impact the quality of the library. If stretches of randomization are below 15 codons and you have about 5-6 of those the library quality should be above 80%.


 

Q- How about an actual estimate of the price? $500, $5000, $50000 per site?

A- It depends on the deliverables you are choosing and how many sites you want to address. Let me give you 2 examples. If you would like to receive all single substitution variants in one tube pricing ranges from 100-150 USD per site. If you request the delivery of an average of 16 mutations per site delivered as single constructs on 96 well plates costs will be between 800-1,200 USD per site.


 

Q- What type of structural data is available? Is this only with comparison to current knowledge, or can one compare changes in aa residues to changes in structure?

A- The influence of an amino acid mutation on protein structure is difficult to predict for most proteins. This is the reason why directed evolution strategies are so powerful. Still, it is a good idea to take existing knowledge on structure into account. For example, many enzymes loose function if changes are introduced within the hydrophobic core of the protein. On the other hand, improving mutations do not necessarily have to be in the vincinity of the active site but may be located on the opposite side of the enzyme, apparently having an unpredictable steric effect on the active site.



Q- I'm confused- is the TRIM technology any different than having oligos constructed with the mixtures of trinucleotides? We have had this done with synthetic codons from Glen research.

A- The TRIM technology uses the incorporation of trinucleotides as you explained. We are using very sophisticated technologies however to fuse randomized regions into the constant backbone of the library guaranteeing a maximum amount of correct specimen.



Q- is it possible to control single amino acid mutations in order to study 'KO' mutations of the protein?

A- Yes, this can be done. If you look at the results of site saturation mutagenesis (SSM) libraries where only single substitutions are incorporated into protein, about 1/3 of the mutations destroy the functionality of the protein.



Q- The customization is combinatorial? I lost connection and am not sure if I missed how you are able to customize specific amino acid at a specific position?

A- The customized randomization will be achieved by introducing triplets, each coding for one amino acid only, instead of just single bases during the synthesis process. For each position triplets can be premixed. In that way you have the total control over the appearance and ratio of occurring amino acids.



Q- Are libraries available in Gateway® expression constructs?

A- Libraries can be cloned in any type of vector as long as it is suitable for high-throughput cloning. Libraries can also be cloned into Gateway® vectors.



Q- Do you offer codon optimization? If so, how? Are you good at it? References?

A- Yes, we offer codon optimization with very good results. We recently published a paper covering this topic. It is available for free on this web page



Q- Can different length mutagenic regions be incorporated together into a single library of fragments (for a single order?)

A- Yes, length polymorphisms can be created. Very prominent examples are the length polymorphisms of antibody CDR3. Basically any length increment can be created (plus one codon, plus two codons etc.). However, maximum recommended length of a randomized stretch is 20 adjacent codons).



Q- What organisms are you able to transfect into for specialized customer vectors? Is it only E. coli and B. subtilis?

A- We usually do this for E. coli. If customers provide us with their bacillus strains we also transform bacillus cells. We also use shuttle vectors for all diferent cell types. However, we do not transform yeast, and the actual transformation service is currently limited to E. coli and in some cases bacillus.

Q- Are you able to ensure that no wild-type proteins sequence remains after mutagenesis?

A- Usually yes. Since everything is synthesized de novo, no surplus wildtype is present at any step. It may be, however, that the design of the library itself allows the occurrence of specimen coding for the wildtype protein. Generally that is not a bad idea. It is a good guideline to design the diversity of a library in a way that a small fraction of it is wildtype and the diversity--the sequence space--emanates from this wildtype. This assures that not all specimens will be loss-of-function due to too many mutations.

Q- Can a screen of a library following the mutation be done for a decrease in protein activity? If one is looking for a null mutant versus an improvement?

A- Yes, this can be done. If you look at the results of Site Saturation Mutagenesis libraries where only single substitutions are incorporated into protein about 1/3 of the mutations destroy the functionality of the protein.