Multiplex Automated Genome Engineering (MAGE) for large-scale programming and evolution of cells.

1. Multiplex Automated Genome Engineering (MAGE) for large-scale programming and evolution of cells. 报告人：陈艳

2. 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

3. 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

4. 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

7. 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.

10. 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

11. MAGE产生序列差异的程度是由三个因素共同决定：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。

12. 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.

14. 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