Method for expressing FAEES by recombinant algae and in-vivo bio diesel production

A technology of biodiesel and algae, applied in the fields of bioenergy and genetic engineering

Inactive Publication Date: 2010-11-24
湖北汇特生物医药技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention combines genetic engineering technology with transesterification technology to prepare biodiesel

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment one: algal seed cultivation

[0015] Prepare the diluted LB culture medium for the bought algae species, and spread them in a 1000mL Erlenmeyer flask according to the ratio of algae liquid to culture liquid 1:2. The triangular flask was cultured in a light incubator, and the culture conditions were controlled: the light intensity was 75.3-80.1 mmol / ms, the temperature was 26°C, and the pH was 6-8. Stir three times a day for 1 minute each time. After the algal body reaches a certain density, expand it in a 3000ml Erlenmeyer flask at a ratio of 1:4 between the algae liquid and the culture liquid, and place it in a place with low temperature and weak light. Enter the filtered air for cultivation, and expand the seeds every 3 weeks or so. The procedure for expanding the culture is 3000mL Erlenmeyer flask → 20L narrow-mouth glass bottle → culture in barrel → production pool.

Embodiment 2

[0016] Embodiment two: FAEES gene is cloned into shuttle vector

[0017] The target gene FAEES is connected with the PET28 vector and exists in the form of a plasmid, referred to as PET-FAEES. PCR was performed using PET-FAEES as a template. To clone the FAEES gene in PCR, synthesize the following primers and use the FAEES gene amplification primers:

[0018] (a-1) 5'-ccgCTCGAG ATA GCC ACC ATGCCGCCCTACACC-3'

[0019] (b-1) 5'-tgcTCTAGATCACTGTTTCCCGTTGCCATT-3'

[0020] Primer (a-1) contains an Xho restriction site, and primer (b-1) contains an Xba restriction site.

[0021] PCR (Polymerase Chain Reaction) was performed under the following conditions using "DNA thermal cycler" and Tag DNA polymerase (Takara Co.) as reaction reagents. Reactant:

[0022] PCR buffer, 1.25mM dNTP mix, template, primers (upstream and downstream), Tag enzyme, sterile water. The above components were mixed, and the mixture was subjected to a PCR reaction.

[0023] PCR cycles:

[0024] Denaturat...

Embodiment 3

[0030] Embodiment three: cultivation and transformation of Agrobacterium tumefaciens

[0031] The FAEES-carrier plasmid constructed above was transformed into Agrobacterium tumefaciens competent cells by freeze-thaw method, and spread on solid medium (LB+40mg / L rifampicin+25mg / L streptomycin+75mg / L gentamicin), pick a single colony, in 2mL Agrobacterium liquid medium, cultivate overnight at 28°C, take more than 1mL of the culture, add it to 50mL Agrobacterium liquid medium, and cultivate to OD at 28°C 600 = 1.0, 50mL of Agrobacterium liquid was centrifuged at 3500-4000r / min for 8min to collect the bacteria, resuspended in UM1 liquid medium, and continued to culture at 28°C for 3-4h until OD 600 = 0.5.

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Abstract

The invention belongs to the technical field of biological energy and genetic engineering, in particular relates to algae grease and a method for producing biodiesel thereby. The novel method for expressing FAEES by recombinant algae and producing in-vivo bio diesel comprises a method for preparing the biodiesel by using grease in algae body and (external source) ethanol to perform ester exchange reaction, namely the algae organisms are soaked in a culture medium containing ethanol, FAEES gene is induced into a gene group in the algae body by using agrobacterium rhizogenes as medium for expressing, and the FAEES produced in the algae body catalyzes fatty acid of the algae body per se to react with externally added (external source) ethanol under certain conditions to obtain the biodiesel and the side product glycerol. The method has mild technical condition, low cost and easy control, and has excellent biological industrial application prospect by using in-vivo grease in the algae organisms and in-vitro ethanol.

Description

technical field [0001] The invention belongs to the technical fields of bioenergy and genetic engineering, and particularly relates to algae oil and a method for preparing biodiesel. Specifically, the present invention relates to a method for producing biodiesel by using recombinant algae, which is a biodiesel method with low input and high yield. Using a kind of algae, a gene that can express the activity of fatty acid ethyl ester synthase (hereinafter referred to as FAEES) is transferred into it through genetic engineering technology, and the FAEES expressed in the algae catalyzes the fatty acid in the algae body and the outside world under certain conditions. The added (exogenous) ethanol reacts to produce biodiesel in vivo. Or crush the algae containing the FAEES gene and stably expressing it, separate the algal cells and collect the culture mixture, then add different amounts of (exogenous) ethanol, react at 37°C for 1-24 hours, and perform the in vitro biotransesterific...

Claims

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Application Information

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IPC IPC(8): C10L1/02
CPCY02E50/13Y02E50/10
Inventor 黄赤夫
Owner 湖北汇特生物医药技术有限公司
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