| Chapter title |
A Novel Platform for High-Throughput Gene Synthesis to Maximize Recombinant Expression in Escherichia coli.
|
|---|---|
| Chapter number | 7 |
| Book title |
PCR
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-7060-5_7 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7059-9, 978-1-4939-7060-5
|
| Authors |
Sequeira, Ana Filipa, Brás, Joana L A, Fernandes, Vânia O, Guerreiro, Catarina I P D, Vincentelli, Renaud, Fontes, Carlos M G A, Ana Filipa Sequeira, Joana L. A. Brás, Vânia O. Fernandes, Catarina I. P. D. Guerreiro, Renaud Vincentelli, Carlos M. G. A. Fontes |
| Editors |
Lucília Domingues |
| Abstract |
Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. Here, we describe a high-throughput platform to design and produce multiple synthetic genes (<500 bp) for recombinant expression in Escherichia coli. This pipeline includes an innovative codon optimization algorithm that designs DNA sequences to maximize heterologous protein production in different hosts. The platform is based on a simple gene synthesis method that uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides. This technology incorporates an accurate, automated and cost-effective ligase-independent cloning step to directly integrate the synthetic genes into an effective E. coli expression vector. High-throughput production of synthetic genes is of increasing relevance to allow exploring the biological function of the extensive genomic and meta-genomic information currently available from various sources. |
X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Portugal | 1 | 50% |
| Unknown | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 21 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 7 | 33% |
| Student > Ph. D. Student | 4 | 19% |
| Student > Bachelor | 3 | 14% |
| Librarian | 1 | 5% |
| Student > Doctoral Student | 1 | 5% |
| Other | 1 | 5% |
| Unknown | 4 | 19% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 8 | 38% |
| Agricultural and Biological Sciences | 5 | 24% |
| Social Sciences | 1 | 5% |
| Chemistry | 1 | 5% |
| Unknown | 6 | 29% |