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No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology.

Azizi A, Lam W, Phenix H, Tepliakova L, Roney IJ, Jedrysiak D, Power A, Gupta V, Elnour N, Hanzel M, Tzahristos AC, Sarwar S, Kærn M - J Biol Eng (2015)

Bottom Line: The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes.To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success.Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Institute of Systems Biology, 451 Smyth Road, K1H 8M5 Ottawa, Ontario Canada ; Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, K1H 8M5 Ottawa, Ontario Canada.

ABSTRACT
The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes. Alnahhas et al. (Journal of Biological Engineering 2014, 8:28) recently argued that these constraints should be revised because they impose an unnecessary burden on contributors that use homology-based assembly. To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success. Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.

No MeSH data available.


Related in: MedlinePlus

Charts illustrating the overall success rate and the success rates for each assembly method under different conditions. Success is defined as a 95 % hypergeometric probability or higher that at least one of three clones screened carry a fully functional plasmid. The fraction of failed tests is indicated in grey. The fraction of successful tests is subdivided into different colors to indicate the method used. a All tests. Overall success rate: 65 %. Individual method success rates: 44/56/73/81 % for HR alone, PCR, Seamless and Gibson, respectively. b Tests with 20 ng transformed DNA. Overall: 75 %. Methods: 65/75/83/79 %. c Tests with 20 ng transformed DNA and long regions of overlap between DNA fragments. Overall: 87 %. Methods: 75/83/92/100 %. d Tests performed by A.A. Overall: 69 %. Methods: 61/50/88/75 %. e Tests performed by H.P. Overall: 81 %. Methods: 75/100/62/88 %. f Tests performed by L.T. Overall: 75 %. Methods: 50 %/75 %/100 %/75 %
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Fig2: Charts illustrating the overall success rate and the success rates for each assembly method under different conditions. Success is defined as a 95 % hypergeometric probability or higher that at least one of three clones screened carry a fully functional plasmid. The fraction of failed tests is indicated in grey. The fraction of successful tests is subdivided into different colors to indicate the method used. a All tests. Overall success rate: 65 %. Individual method success rates: 44/56/73/81 % for HR alone, PCR, Seamless and Gibson, respectively. b Tests with 20 ng transformed DNA. Overall: 75 %. Methods: 65/75/83/79 %. c Tests with 20 ng transformed DNA and long regions of overlap between DNA fragments. Overall: 87 %. Methods: 75/83/92/100 %. d Tests performed by A.A. Overall: 69 %. Methods: 61/50/88/75 %. e Tests performed by H.P. Overall: 81 %. Methods: 75/100/62/88 %. f Tests performed by L.T. Overall: 75 %. Methods: 50 %/75 %/100 %/75 %

Mentions: While the four methods worked remarkably well, the screening of up to 20 colonies per assembly can be both expensive and impractical. For this reason, we assessed how well the methods work when fewer colonies are screened. To do this, we calculated for each test the probability that screening three colonies would give at least one colony with a fully functional plasmid, and judged a test as successful if this probability was 95 % or higher. We then counted for each method the fraction of successful tests, and used these fractions to calculate overall success rates as well as to compare the four methods. With this more restrictive measure of assembly success, we found that 64 % of the 192 tests were successful (Fig. 2a). While HR alone was successful in 44 % of the tests that used this method, the tests that used PCR, Seamless or Gibson assembly had success rates of 56 %, 73 % or 81 %, respectively.Fig. 2


No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology.

Azizi A, Lam W, Phenix H, Tepliakova L, Roney IJ, Jedrysiak D, Power A, Gupta V, Elnour N, Hanzel M, Tzahristos AC, Sarwar S, Kærn M - J Biol Eng (2015)

Charts illustrating the overall success rate and the success rates for each assembly method under different conditions. Success is defined as a 95 % hypergeometric probability or higher that at least one of three clones screened carry a fully functional plasmid. The fraction of failed tests is indicated in grey. The fraction of successful tests is subdivided into different colors to indicate the method used. a All tests. Overall success rate: 65 %. Individual method success rates: 44/56/73/81 % for HR alone, PCR, Seamless and Gibson, respectively. b Tests with 20 ng transformed DNA. Overall: 75 %. Methods: 65/75/83/79 %. c Tests with 20 ng transformed DNA and long regions of overlap between DNA fragments. Overall: 87 %. Methods: 75/83/92/100 %. d Tests performed by A.A. Overall: 69 %. Methods: 61/50/88/75 %. e Tests performed by H.P. Overall: 81 %. Methods: 75/100/62/88 %. f Tests performed by L.T. Overall: 75 %. Methods: 50 %/75 %/100 %/75 %
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4464999&req=5

Fig2: Charts illustrating the overall success rate and the success rates for each assembly method under different conditions. Success is defined as a 95 % hypergeometric probability or higher that at least one of three clones screened carry a fully functional plasmid. The fraction of failed tests is indicated in grey. The fraction of successful tests is subdivided into different colors to indicate the method used. a All tests. Overall success rate: 65 %. Individual method success rates: 44/56/73/81 % for HR alone, PCR, Seamless and Gibson, respectively. b Tests with 20 ng transformed DNA. Overall: 75 %. Methods: 65/75/83/79 %. c Tests with 20 ng transformed DNA and long regions of overlap between DNA fragments. Overall: 87 %. Methods: 75/83/92/100 %. d Tests performed by A.A. Overall: 69 %. Methods: 61/50/88/75 %. e Tests performed by H.P. Overall: 81 %. Methods: 75/100/62/88 %. f Tests performed by L.T. Overall: 75 %. Methods: 50 %/75 %/100 %/75 %
Mentions: While the four methods worked remarkably well, the screening of up to 20 colonies per assembly can be both expensive and impractical. For this reason, we assessed how well the methods work when fewer colonies are screened. To do this, we calculated for each test the probability that screening three colonies would give at least one colony with a fully functional plasmid, and judged a test as successful if this probability was 95 % or higher. We then counted for each method the fraction of successful tests, and used these fractions to calculate overall success rates as well as to compare the four methods. With this more restrictive measure of assembly success, we found that 64 % of the 192 tests were successful (Fig. 2a). While HR alone was successful in 44 % of the tests that used this method, the tests that used PCR, Seamless or Gibson assembly had success rates of 56 %, 73 % or 81 %, respectively.Fig. 2

Bottom Line: The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes.To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success.Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Institute of Systems Biology, 451 Smyth Road, K1H 8M5 Ottawa, Ontario Canada ; Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, K1H 8M5 Ottawa, Ontario Canada.

ABSTRACT
The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes. Alnahhas et al. (Journal of Biological Engineering 2014, 8:28) recently argued that these constraints should be revised because they impose an unnecessary burden on contributors that use homology-based assembly. To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success. Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.

No MeSH data available.


Related in: MedlinePlus