In vitro template-change PCR to create single crossover libraries: a case study with B. thuringiensis Cry2A toxins.
During evolution the creation of single crossover chimeras between duplicated paralogous genes is a known process for increasing diversity. Comparing the properties of homologously recombined chimeras with one or two crossovers is also an efficient strategy for analyzing relationships between sequence variation and function. However, no well-developed in vitro method has been established to create single-crossover libraries. Here we present an in vitro template-change polymerase change reaction that has been developed to enable the production of such libraries. We applied the method to two closely related toxin genes from B. thuringiensis and created chimeras with differing properties that can help us understand how these toxins are able to differentiate between insect species.
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Changlong
C
Shu
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Jianqiao
J
Zhou
School of Life Sciences, University of Sussex, Falmer, Brighton, UK.
Neil
N
Crickmore
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Xianchun
X
Li
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Fuping
F
Song
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Gemei
G
Liang
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Kanglai
K
He
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Dafang
D
Huang
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China.
Jie
J
Zhang
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002678:Chimera; D003434:Crossing Over, Genetic; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis