1 Module 2 overview lecture lab 1 Introduction to the module 1 Start up protein eng 2 Rational protein design 2 Site directed mutagenesis 3 Fluorescence and sensors 3 DNA ampli cation 4 Protein expression 4 Prepare expression system SPRING BREAK 5 Review gene analysis 5 Gene analysis induction 6 Puri cation and protein analysis 6 Characterize expression 7 Binding af nity measurements 7 Assay protein behavior 8 High throughput engineering 8 Data analysis 2 Lecture 5 Review gene analysis I Review of the project A Project aims and rationale B Methods work completed so far II Analysis of mutant genes A Restriction digests B DNA sequencing 3 Module 2 assignment Protein engineering research article 1 Abstract 2 Introduction 3 Materials and Methods 4 Results 5 Discussion 6 References 7 Figures 4 Module 2 assignment Protein engineering research article 1 Abstract 2 Introduction Why are calcium sensors important What is protein engineering how does it relate What is inverse pericam Why is it useful interesting to tune pericam Why did you choose your mutations 3 4 5 6 7 Materials and Methods Results Discussion References Figures 5 Module 2 assignment Protein engineering research article 1 Abstract 2 Introduction Why are calcium sensors important What is protein engineering how does it relate What is inverse pericam Why is it useful interesting to tune pericam Why did you choose your mutations 3 4 5 6 7 Materials and Methods Results Discussion References Figures 6 Restriction enzymes digest speci c DNA sequences www wikipedia com Wikipedia source http en wikipedia org wiki Restriction enzyme License CC BY SA This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse you designed mutations that can be assessed by restriction mapping TACATCAGCGCTGCTCAG ATGTAGTCGCGACGAGTC Y I S A A Q TACATCCTCGCTGCGCAG ATGTAGGAGCGACGCGTC Y I L A A Q How do restriction endonucleases work BamHI 7 Reprinted by permission from Macmillan Publishers Ltd Nature Structural Biology Source Viadiu H and A K Aggarwal The Role of Metals in Catalysis by the Restriction Endonuclease Bam HI Nature Structural Biology 5 1998 910 916 1998 Courtesy of Elsevier Inc http www sciencedirect com Used with permission Source Viadiu H and A K Aggarwal Structure of BamHI Bound to Nonspecific DNA A Model for DNA Sliding Molecular Cell 5 no 5 2000 889 895 Viadiu Aggarwal 1998 2000 8 FspI diagnostic digest std ctr restriction endonucleases in cloning mut ligation Courtesy of Life Technologies Used with permission Genetic polymorphisms can be associated with different distributions of restriction sites restriction fragment length polymorphisms RFLPs used for genotyping Inheritance of RFLP markers Parents A 1 2 3 a Siblings B b AA aa Aa Aa Aa aa Aa AA Genotypes Figure by MIT OpenCourseWare Suppose alleles A and B each occur in 50 of the population and segregated independently what are the chances that a randomly chosen individual displays the AB phenotype How many biallelic polymorphisms would have to be considered for each genotype to have a 1 1 000 000 chance of occurring assuming equal prevalence of each 9 10 Reprinted by permission from Macmillan Publishers Ltd Nature Reviews Genetics Source Jobling M A and P Gill Encoded evidence DNA in Forensic Analysis Nature Reviews Genetics 5 October 2004 739 751 2004 Jobling 2004 Nat Rev Genetics 11 How does sequencing work Perform PCR on template to be sequences each PCR reaction is terminated by a nucleotide analog that can be incorporated but not added to Terminated PCR products must be labeled in some way nucleotides linked by phosphodiester bonds NH2 dATP O N O N O O P O O H O N H O P NH2 P O O N P O O P O O OH N N H O H H OH H H NH2 H H O O NH2 N H O H O N P N O O N H O H O NH N H O P O H H O NH H HO H O H H H Public domain image N N H H O O ddATP O H O www wikipedia com N O O O N N O NH2 O P O O O P O N O O P O O OH H H H H H N 12 sequencing with radioactive ddNTPs run products in four separate lanes on gel expose X ray lm longer fragments template sequence shorter fragments Wikipedia Public domain image one pot sequencing more common today 13 www wikipedia com Public domain images MIT OpenCourseWare http ocw mit edu 20 109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use visit http ocw mit edu terms