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Construction and Electroporation of luxAB Expression Vector of Spirulina Platensis

Author: ZhuYiWei
Tutor: TangXinZuo
School: Anhui Agricultural University
Course: Microbiology
Keywords: Spirulina platensis vector construction luxAB gene
CLC: Q943.2
Type: Master's thesis
Year: 2009
Downloads: 33
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Abstract


The aim of this experiment is to set up and to perfect the genetic system of Spirulina. We studied the extent that the Spirulina A9 strain is sensitive to Amp, and cloned the luxAB gene fragment and got the vector fragment with promoter Ubil and amp gene. With the two fragments, we constructed the new plasmid vector. The aim of the pretreatment of Spirulina cells is to restrain the activity of nuclease, and then the electroporation was used to transformate the Spirulina cells.The Spirulina A9 strain is relatively sensitive to Amp. The minimum inhibitory concentration (MIC) of Amp is 100μg·ml-1 in liquid situation and 200μg·ml-1 in solid situation Amp might be suitable selection genetic markers for the Spirulina, and the selection concentrations is designed as 200μg·ml-1.In this experiment, the plasmid pUC?GUS was digested by the restriction enzyme SmaI and EcoRI, and the vector fragment with promoter Ubil and amp gene was recovered. According to the sequence of luxAB gene in the plasmid pRL1063a, primers were designed with the digested points of SmaI and EcoRI in the upriver and downriver. With the template of pRL1063a digested by SalI, the luxAB gene fragment was cloned by PCR. Then the victor fragments and luxAB gene were recombined with T4 DNA ligase. The reconstructed plasmid is named as pUC?luxAB with the luxAB and amp gene. Furthermore, the competence cell of E. coli MC4100A strain was prepared with CaCl2 and the reconstructed plasmid was transformed into E.coli MC4100A.We cultivated E.coli MC4100A cells to replicate the reconstructed plasmid. Digestion identified the reconstructed plasmid. The reconstructed plasmid pUC?luxAB was transformed and the expression result identified with fat aldehyde.In this experiment, we got the vector fragment with promoter Ubil and amp gene was obtained and the luxAB gene fragment was amplified by PCR. The victor fragments and luxAB gene were recombined with T4 DNA ligase. The reconstructed plasmid pUC?luxAB was proved by enzyme digestion.We cultured A9 strain at 24℃and treated with 2 mmol·L-1 EDTA for 24h before transformation, and useed ultrasonic treatment with power of 300w for 70s. Reconstructed plasmid was used to transformate Spirulina A9 cells using electroporation.In this experiment, the time of electroporation is 5.0ms and the intension of electric filed is 4kv·cm-1. The electroporation buffer is 1.0 mmol·L-1HEPES (pH7.5) + 0.5 mol·L-1 sucrose, and the electroporation object is Spirulina A9 cells that are in medium-term of logarithm growth.

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CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
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