Download - Multiplex Automated Genome Engineering (MAGE) for large-scale programming and evolution of cells
Multiplex Automated Genome Engineering (MAGE) for large-scale programming and evolution of cells.
报告人:陈艳
• in vivo methods such as recombination-based genetic engineering (recombineering) have enabled efficient modification of single genetic targets using single-stranded DNA (ssDNA)
• no such attempts have been made to modify genomes on a large and parallel scale.
MAGE
• 多基因工程 (MAGE)• MAGE simultaneously targets many locations on the
chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity.
• the process is cyclical and scalable• MAGE technology to facilitate rapid and continuous generation
of a diverse set of genetic changes (mismatches, insertions, deletions).
• 单基因工程只能改良生物的单基因性状,不能改变高等生物的多基因性状
• 而多基因工程非但能改良生物品种而且能创造生物新物种
According to “High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides.” by Ellis, H. M et. from PNAS
λ Red-mediated ssDNA recombination provides a rapid and highly efficient approach for generating recombinant DNA
molecules. ------ssDNA Recombination Requires Only Beta.
与双链 DNA重组机制不同 : Red 重组系统由 λ 噬菌体 exo 、 bet 、 gam 三个基因分别编码的三种蛋白组成 : Exo 蛋白是一种核酸外切酶 ,结合在双链 DNA 的末端 , 从5′端向 3′端降解 DNA , 产生 3′突出端 ; Beta 蛋白结合在单链 DNA
上 ,介导互补单链 DNA退火 ; Gam 蛋白可与 RecBCD酶结合 ,抑制其降解外源 DNA的活性。
Red同源重组技术具有同源序列短 (40 ~ 60 bp) 、重组效率高的特点。 这种技术可在 DNA靶标分子的任意位点进行基因敲除、敲入、点突变
等操作 ,无需使用限制性内切酶和连接酶
λ Red-mediated ssDNA recombination provides a rapid and highly efficient approach for generating recombinant DNA molecules. Because this process is highly efficient, one can directly screen for mutations in the absence of selection.
Oligo-mediated recombination occurs in both E. coli and yeast, suggesting that recombination via annealing of ssDNAs may occur in a wide range of organisms. Thus, recombineering with ssDNA may be applicable to higher eukaryotes as well, perhaps by a mechanism as simple as overexpressing Beta or a functionally similar ssDNA annealing protein, or more directly by introducing the protein itself into the eukaryotic cell during electroporation.
determine the rate at which MAGE generates sequence diversity :use three different 90-mer oligos to produce mismatch changes in a targeted region of the lacZ gene in three distinct cell populations --- (基于蓝白筛选原理、 DNA 测序)
经过连续的MAGE周期, LacZ基因的序列与野生型的差异越来越大
• MAGE 产生序列差异的程度是由三个因素共同决定:
• (1) the degree of sequence variation desired at each locus;
• (2) the number of loci targeted; • (3) the number of MAGE cycles performed. 无论细胞群体数量多少,可以通过计算预测和MAGE连续循环生成所有的变种。
• The device executed repeatedly and continuously through the following steps:
• (1) grow cells at 30 to a pre-set density (that is, OD600 nm ℃of 0.7);
• (2)induce cells for allelic replacement via 42 heat shock for ℃15min;
• (3) chill cells at 4 to halt cellular metabolism; ℃• (4) wash cells through 15–20 iterations of filtration and
resuspension with dH2O; • (5) mix cell suspension and synthetic DNA; • (6) deliver DNA into cells by electroporation;• (7) resuspend electroporated cells with growth media.
MAGE 的应用 通过优化含有 pAC-LYC 质粒大肠杆菌( EcHW2 )的
DXP 生物合成途径的代谢通量来过量生产番茄红素。 用 oligos 的核糖体结合位点( RBS )的简并序列(DDRRRRRDDDD; D=A,G,T; R=A,G) (设计成类似Shine–Dalgarno 序列( TAAGGAGGT ))来替换 20 个内源靶基因 (dxs, dxr, ispD, ispE, ispG, ispH, idi, ispA, appY,
rpoS, crl, elbA, elbB, yjiD, purH, rnlA, yggT, ycgZ, ymgA, ariR) ,以提高翻译效率。 分支途径的 4 个基因 (ytjC, fdhF, aceE, gdhA) 因在 oligos的开放阅读框中引入 2 个无义突变而失活,进一步提高DXP 途径的通量。
与单基因操作相比,一次操作能同时优化 24个基因