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 amplification4. Protein expression   4. Prepare expression systemSPRING BREAK5. Review & gene analysis 5. Gene analysis & induction6. Purification and protein analysis 6. Characterize expression7. Binding & affinity 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. Materials and Methods4. Results5. Discussion6. References7. 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. Materials and Methods4. Results5. Discussion6. References7. FiguresRestriction enzymes digest specific DNA sequenceswww.wikipedia.comyou designed mutations that can be assessed by restriction mapping:...TACATCAGCGCTGCTCAG... ...TACATCCTCGCTGCGCAG......ATGTAGTCGCGACGAGTC... ...ATGTAGGAGCGACGCGTC...Y I S A A Q Y I L A A Q6License CC BY-SA. This content is excluded from our Creative Commons license. For more information, see http://ocw.mit.edu/fairuse.© Wikipedia (source: http://en.wikipedia.org/wiki/Restriction_enzyme).7How do restriction endonucleases work?BamHI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. Viadiu & Aggarwal (1998, 2000)"Structure of BamHI Bound to Nonspecific DNA: A Model for DNA Sliding." Molecular Cell 5, no. 5 (2000): 889-895.FspIstd ctr mutdiagnosticdigestrestrictionendonucleasesin cloningligation8Courtesy of Life Technologies. Used with permission.9Genetic polymorphisms can be associated with different distributionsof restriction sites–restriction fragment length polymorphisms(RFLPs) used for genotyping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 consideredfor each genotype to have a 1:1,000,000 chance of occurring,assuming equal prevalence of each?12 3ABabParentsGenotypesSiblingsAA aa Aa Aa AaaaAa AAInheritance of RFLP markersFigure by MIT OpenCourseWare.10Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics. Jobling (2004) Nat. Rev. GeneticsSource: Jobling, M. A., and P. Gill. "Encoded evidence: DNA in Forensic Analysis." Nature Reviews Genetics 5, (October 2004): 739-751. © 2004.11www.wikipedia.com(Public domain image) nucleotides linkedby phosphodiesterdATPNH2NNbondsOOONN-OOP-OPOPO-POOO-O-O--OHONNH2HHNHONHHOHNOH HOPHHHNH2-OONNH2ONHOHONddATPNONHNNONH2NOHHHHHOONHOHOHONOHHOPHH-OOOONN-OPOPO-POOOHH-OHPO-O-O-HHHHHHow 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.!12sequencing with radioactive ddNTPsrun products in four separatelanes on gel, expose X-ray filmlonger fragmentsshorter fragmentsWikipedia (Public domain image) template sequence13“one pot” sequencing more common today:www.wikipedia.com(Public domain images)MIT OpenCourseWarehttp://ocw.mit.edu20.109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use, visit: