我们提出了一个协议为利用高通量测序蛋白的综合单站点饱和突变库功能评估。重要的是,这种方法采用正交引物对复图书馆建设和测序。提供了使用在氨苄青霉素的临床相关剂量选择的TEM-1β内酰胺酶代表性结果。
Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.
诱变早已在实验室用于研究生物系统及其演进的属性,并产生突变蛋白质或微生物与增强的或新的功能。虽然早期的方法在其上产生生物随机突变的方法依赖,重组DNA技术的出现使研究人员能够引入位点特异性方式与DNA选择的变化, 即 ,位点定向诱变1,2。与目前的技 术,一般是在聚合酶链反应(PCR),使用诱变寡核苷酸,它是相对容易的在给定基因3,4-创建并评估突变的小的数字( 例如 ,点突变)。这是更为困难然而,当目标的办法,例如,所有可能的单站点的创建和评估(或更高阶)的突变。
虽然很多已经从早期的研究试图评估大量m的教训在基因utations,所采用的技术往往是费力的,例如要求每个突变评估独立使用的废话抑制5-7株,或在自己的能力量化,由于Sanger测序8的低测序深度有限。在这些研究中所使用的技术已在很大程度上被利用高通量测序技术方法9-12取代。这些概念简单的方法意味着创建包括了大量的突变库,库中经受了功能的屏幕或选择,然后深测序( 即 ,> 10 6排序的顺序读取时)之前获得图书馆,选择后。以这种方式,大量的突变的表型或健身效果,表示为在每个突变体的群体频率的变化,可以同时和更定量评估。
我们之前推出了SIMP( 即 ,全蛋白饱和诱变文库)用于评估蛋白质的所有可能的单氨基酸突变的文库,适用于基因的一个长度比测序长文件的方法读取长度11,13:首先,每个氨基酸位置是随机通过位点定向诱变的PCR。在此过程中,该基因被分成与由测序平台收纳总长度连续位置组成的组。诱变的PCR产物为每个组,然后组合,并且每个基团独立地进行选择和高通量测序。通过保持序列和测序读长在基因突变的位置之间的对应关系,这种方法也有最大化的测序深度的优点:当一个可以简单地序列中短窗这样的库,不要拆开成团( 例如 ,通过一个标准的猎枪。测序方法),最读取得到的将是野生型和由此m个浪费测序吞吐量ajority( 例如,在100个氨基酸(300 bp)的窗户测序一个500个氨基酸的蛋白的一个全蛋白饱和诱变文库,以最低80%的读出将是野生型序列)。
这里,提出了一种协议,它利用高通量测序为全蛋白饱和诱变文库的功能评估,使用上述方法( 图1中所述)。重要的是,我们引入的正交的引物的使用量在库中克隆过程条形码每个序列组,这允许它们被复用到一个库,同时进行筛选或选择,然后解复用为深度测序。由于序列组不进行选择独立,这减少了工作量,并确保各突变经历选择的相同水平。 TEM-1β内酰胺酶,其赋予对高层次性的酶β内酰胺类抗生素( 如氨苄青霉素)的细菌被用作模型系统14-16。的协议是用于TEM-1在大肠杆菌中一个全蛋白饱和诱变文库的评估中描述大肠杆菌下,在对一个临床剂量氨苄青霉素(50微克/毫升)17,18的近似的血清水平的选择。
这里一个协议被用于进行全蛋白饱和诱变文库的功能评估,使用高通量测序技术说明。该方法的一个重要方面是在克隆过程中使用的正交引物。简单地说,每个氨基酸位置由诱变PCR随机,并混合在一起成为其组合序列长度由高通量测序收容位置的基团。这些基团被克隆入含有成对正交引发位点,混合在一起,并进行选择的质粒载体,然后使用正交引物解复用,并随后深测序。由于突变测序中阅读?…
The authors have nothing to disclose.
R.R. acknowledges support from the National Institutes of Health (RO1EY018720-05), the Robert A. Welch Foundation (I-1366), and the Green Center for Systems Biology.
Typtone | Research Products Intl. Corp. | T60060-1000.0 | |
Yeast extract | Research Products Intl. Corp. | Y20020-500.0 | |
Sodium chloride | Fisher Scientific | BP358-212 | |
Potassium chloride | Sigma-Aldrich | P9333-500G | |
Magnesium sulfate | Sigma-Aldrich | M7506-500G | |
Agar | Fisher Scientific | BP1423-500 | |
Tetracycline hydrochloride | Sigma-Aldrich | T7660-5G | |
petri plates | Corning | 351029 | |
MATLAB | Mathworks | http://www.mathworks.com/products/matlab/ | |
Oligonucleotide primers | Integrated DNA Technologies | https://www.idtdna.com/pages/products/dna-rna/custom-dna-oligos | 25 nmol scale, standard desalting |
pBR322_AvrII | available upon request | pBR322 plasmid modified to contain AvrII restriction site downstream of the TEM-1 gene | |
pBR322_OP1 – pBR322_OP5 | available upon request | five modified pBR322 plasmids each containing a pair of orthogonal priming sites | |
Q5 high-fidelity DNA polymerase | New England Biolabs | M0491L | includes 5X PCR buffer and PCR additive (GC enhancer) |
15 mL conical tube | Corning | 430025 | |
Multichannel pipettes (Eppendorf ResearchPlus) | Eppendorf | ||
PCR plate, 96 well | Fisher Scientific | 14230232 | |
96 well plate seal | Excel Scientific | F-96-100 | |
Veriti 96-well thermal cycler | Applied Biosystems | 4375786 | |
6X gel loading dye | New England Biolabs | B7024S | |
Agarose | Research Products Intl. Corp. | 20090-500.0 | |
Ethidium bromide | Bio-Rad | 161-0433 | |
UV transilluminator (FOTO/Analyst ImageTech) | Fotodyne Inc. | http://www.fotodyne.com/content/ImageTech_gel_documentation | |
EB buffer | Qiagen | 19086 | |
96-well black-walled, clear bottom assay plates | Corning | 3651 | |
Lambda phage DNA | New England Biolabs | N3011S | |
PicoGreen dsDNA reagent | Invitrogen | P7581 | dsDNA quantitation reagent, used in protocol step 2.2.4 |
Victor 3V microplate reader | PerkinElmer | ||
DNA purification kit | Zymo Research | D4003 | |
Microcentrifuge tubes | Corning | 3621 | |
Long-wavelength UV illuminator | Fisher Scientific | FBUVLS-80 | |
Agarose gel DNA extraction buffer | Zymo Research | D4001-1-100 | |
AatII | New England Biolabs | R0117S | |
AvrII | New England Biolabs | R0174L | |
T4 DNA ligase | New England Biolabs | M0202S | |
EVB100 electrocompetent E. coli | Avidity | EVB100 | |
Electroporator (E. coli Pulser) | Bio-Rad | 1652102 | |
Electroporation cuvettes | Bio-Rad | 165-2089 | |
Spectrophotometer (Ultrospec 3100 pro) | Amersham Biosciences | 80211237 | |
50 mL conical tubes | Corning | 430828 | |
Plasmid purification kit | Macherey-Nagel | 740588.25 | |
8 well PCR strip tubes | Axygen | 321-10-551 | |
Qubit dsDNA HS assay kit | Invitrogen | Q32854 | dsDNA quantitation reagent |
Qubit assay tubes | Invitrogen | Q32856 | |
Qubit fluorometer | Invitrogen | Q32866 | |
Ampicillin sodium salt | Akron Biotechnology | 50824296 | |
MiSeq reagent kit v2 (500 cycles) | Illumina | MS-102-2003 | |
MiSeq desktop sequencer | Illumina | http://www.illumina.com/systems/miseq.html | alternatively, one could sequence on Illumina HiSeq platform |
FLASh software | John Hopkins University – open source | http://ccb.jhu.edu/software/FLASH/ | software to merge paired-end reads from next-generation sequencing data |
AatII_F | GATAATAATGGTTTCTTAGACGTCAGGTGGC | ||
AvrII_R | CTTCACCTAGGTCCTTTTAAATTAAAAATGAAG | ||
AvrII_F | CTTCATTTTTAATTTAAAAGGACCTAGGTGAAG | ||
AatII_OP1_R | ACCTGACGTCCGTATTTCAACTGTCCGGTCTAAGAAACCATTATTATCATGACATTAAC | ||
AatII_OP2_R | ACCTGACGTCCGCTCACGGAGTGTACTAATTAAGAAACCATTATTATCATGACATTAAC | ||
AatII_OP3_R | ACCTGACGTCGTACGTCTGAACTTGGGACTTAAGAAACCATTATTATCATGACATTAAC | ||
AatII_OP4_R | ACCTGACGTCCCGTTCTCGATACCAAGTGATAAGAAACCATTATTATCATGACATTAAC | ||
AatII_OP5_R | ACCTGACGTCGTCCGTCGGAGTAACAATCTTAAGAAACCATTATTATCATGACATTAAC | ||
OP1_F | GACCGGACAGTTGAAATACG | ||
OP1_R | CGACGTACAGGACAATTTCC | ||
OP2_F | ATTAGTACACTCCGTGAGCG | ||
OP2_R | AGTATTAGGCGTCAAGGTCC | ||
OP3_F | AGTCCCAAGTTCAGACGTAC | ||
OP3_R | GAAAAGTCCCAATGAGTGCC | ||
OP4_F | TCACTTGGTATCGAGAACGG | ||
OP4_R | TATCACGGAAGGACTCAACG | ||
OP5_F | AGATTGTTACTCCGACGGAC | ||
OP5_R | TATAACAGGCTGCTGAGACC | ||
Group1_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGCATTTTGCCTACCGGTTTTTGC | ||
Group1_R | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNTCTTGCCCGGCGTCAAC | ||
Group2_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGAACGTTTTCCAATGATGAGCAC | ||
Group2_R | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGTCCTCCGATCGTTGTCAGAAG | ||
Group3_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNAGTAAGAGAATTATGCAGTGCTGCC | ||
Group3_R | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNTCGCCAGTTAATAGTTTGCGC | ||
Group4_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCCAAACGACGAGCGTGACAC | ||
Group4_R | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGCAATGATACCGCGAGACCC | ||
Group5_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCGGCTGGCTGGTTTATTGC | ||
Group5_R | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNTATATGAGTAAACTTGGTCTGACAG | ||
501_F | AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGAC | ||
502_F | AATGATACGGCGACCACCGAGATCTACACATAGAGGCACACTCTTTCCCTACACGAC | ||
503_F | AATGATACGGCGACCACCGAGATCTACACCCTATCCTACACTCTTTCCCTACACGAC | ||
504_F | AATGATACGGCGACCACCGAGATCTACACGGCTCTGAACACTCTTTCCCTACACGAC | ||
505_F | AATGATACGGCGACCACCGAGATCTACACAGGCGAAGACACTCTTTCCCTACACGAC | ||
701_R | CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTG | ||
702_R | CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTG |