Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

System for capturing and modifying large pieces of genomic DNA and constructing organisms with synthetic chloroplasts

An organism and chloroplast technology, applied in recombinant DNA technology, the use of vectors to introduce foreign genetic material, sugar derivatives, etc., can solve problems such as carrier limitation, increasing the complexity of genetically engineered plants and algae, and complex chloroplast translation.

Inactive Publication Date: 2011-01-05
SAPPHIRE ENERGY
View PDF11 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these vectors are limited by their small insertion capacity
A second type of vector (also known as a fragmented vector) has recombinogenic sequences but cannot transfer the recovered insert DNA to bacteria for mass production of DNA
For example, although chloroplasts are thought to have originated from the endosymbiosis of photosynthetic bacteria into eukaryotic hosts, the translation of chloroplasts is more complex
The presence of multiple chloroplasts with multi-copy chloroplast genomes increases the complexity of genetically engineered plants and algae

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • System for capturing and modifying large pieces of genomic DNA and constructing organisms with synthetic chloroplasts
  • System for capturing and modifying large pieces of genomic DNA and constructing organisms with synthetic chloroplasts
  • System for capturing and modifying large pieces of genomic DNA and constructing organisms with synthetic chloroplasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1: DNA purification and analysis

[0146] DNA is isolated and analyzed according to methods known in the art.

[0147] To prepare DNA from Chlamydomonas reinhardtii for use as a template for PCR, 10 6 Algal cells (from agar plates or liquid media) were suspended in 10 mM EDTA and heated to 95°C for 10 minutes, then cooled to approximately 23°C. This solution was added directly to the PCR mix.

[0148] To prepare purified chloroplast DNA from Chlamydomonas reinhardtii, 5 × 10 8 Algae cells were washed once with water, centrifuged at 3000×g for 10 min, and resuspended in 10 mL of lysing solution (10 mM Tris pH=8.0, 10 mM EDTA, 150 mM NaCl, 2% SDS, 2% sodium lauryl creatine and 25ug / mL pronase (Roche)) and incubated at 37°C for 1 hour. The lysate was then gradually extracted with phenol / chloroform, followed by two chloroform washes. Total DNA was isolated by ethanol precipitation and resuspension in resuspension buffer (10 mM Tris pH=7.4, 1 mM EDTA and 0.1 m...

Embodiment 2

[0155] Embodiment 2: transformation method

[0156] E. coli strain DH10B or Genehog were prepared as electrocompetent cells by the following steps: Cells were grown to an OD of 0.7 600 , then collected and washed twice with ice-cold 10% glycerol, snap-frozen in a dry-ice ethanol bath and kept at -80°C. All yeast or algal DNA was prepared in a Bio-Rad Gene Pulsar Electroporator using, for example, a 0.1 cm cuvette at 1,800 V, 200 ohms and 25 mF and electroporated into E. coli. Cells were recovered and clones were selected in agar growth medium containing one or more antibiotics such as kanamycin (50 ug / mL), ampicillin (100 ug / mL), gentamicin (50 ug / mL ), tetracycline (51ug / mL) or chloramphenicol (34ug / mL).

[0157] Yeast strains YPH857, YPH858 or AB1380 can be transformed by methods such as Sheistl & Geitz (Curr. Genet. 16:339346, 1989) and Sherman et al., "Laboratory Course Manual Methods in Yeast Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986), ...

Embodiment 3

[0160] Example 3: A heterozygous gap-filling vector capturing the chloroplast genome

[0161] In this example, a system was established using a heterozygous gap-filler vector that captures chloroplast DNA (Fig. 1). The hybrid gap-filling vector backbone contains yeast elements allowing it to function as a yeast artificial plasmid (YAP) and bacterial elements allowing it to function as a plasmid artificial chromosome (PAC). Yeast elements include yeast selectable marker sequences (such as TRP1 or LEU2), yeast centromere sequences (CEN), and yeast autonomously replicating nucleotide sequences (ARS). Bacterial elements include P1 or bacterial origin of replication sequences and bacterial selectable marker sequences such as Kanr.

[0162] To manipulate the hybrid gap-filling vector, the vector pDOCI (SEQ ID NO. 1) was generated. Part of pTRP-AU ( figure 2 ) is amplified using a PCR primer pair that anneals to sites surrounding the region comprising TEL, ADE2 and URA3. One p...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The functional analysis of genes frequently requires the manipulation of large genomic regions. A yeast-bacteria shuttle vector is described that can be used to clone large regions of DNA by homologous recombination. Also described is a method for isolating entire genomes, including chloroplast genomes, or large portions thereof, and manipulating the same. Also described are methods for determining minimal genomes, minimal pathway requirements, and minimal organelle genomes.

Description

[0001] cross reference [0002] This application claims priority to and benefit of US Provisional Application No. 60 / 978,024 (filed October 5, 2007), which application is incorporated herein by reference. Background technique [0003] For the functional analysis of many genes, researchers need to isolate and manipulate large DNA fragments. The advent of genomics and the study of genomic regions of DNA has created a need for vectors capable of carrying large regions of DNA. [0004] Generally, two types of yeast vector systems are currently available. The first type of vector is one capable of transferring small inserts of DNA between yeast and bacteria. The second type of vector is a fragmented vector that produces intermediate or terminal deletions in yeast artificial chromosomes (YACs). Small insertion shuttle vectors are able to recombine with and recover homologous sequences. They are centromere-based and replicate stably and autonomously in yeast, but also contain hig...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/00C12N15/09C07H21/04C07K14/52
CPCC12N15/81C12N15/79C12N15/82
Inventor M·门德斯B·奥奈尔K·米克尔森
Owner SAPPHIRE ENERGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com