Observations of Observations of Multiple Multiple Transformation KitsTransformation KitsBryan Smith (LaLumiere School) Bryan Smith (LaLumiere School) Teresa Pairitz (Marian High School)Teresa Pairitz (Marian High School)Shelly Gregory (South Bend Adams High School)Shelly Gregory (South Bend Adams High School)ND RET Molecular Biology Workshop 2009ND RET Molecular Biology Workshop 2009

TransformationTransformation

Occurs when a cell takes up and expresses a new Occurs when a cell takes up and expresses a new piece of DNA (plasmid) which it did not previously havepiece of DNA (plasmid) which it did not previously have

Stumbled upon Stumbled upon initially by F. initially by F. Griffith Griffith (1928) while (1928) while studying studying pneumonia in pneumonia in micemice

Uses of TransformationUses of Transformation

GMOs—to deter from frost, pests; increase drought resistance; GMOs—to deter from frost, pests; increase drought resistance; boost nutrient content; boost nutrient content; potential for crops to deliver potential for crops to deliver vaccines for infectious diseasesvaccines for infectious diseases

Bioremediation—oil-digesting bacteriaBioremediation—oil-digesting bacteria

Medicine—human insulin, clotting Medicine—human insulin, clotting factor, growth hormone production factor, growth hormone production by bacteria; target and destroy by bacteria; target and destroy hard-to-reach cancer cells (3/2009)hard-to-reach cancer cells (3/2009)

Basic Transformation Basic Transformation Procedure Common to the 3 Procedure Common to the 3 KitsKits

Bacteria and plasmid chilled on ice (4 C) Bacteria and plasmid chilled on ice (4 C) in CaClin CaCl22 to allow cell membrane to allow cell membrane permeability to plasmidpermeability to plasmid

Heat shock (42 C), times variable, to Heat shock (42 C), times variable, to improve plasmid permeabilityimprove plasmid permeability

Ice, times variableIce, times variable Recovery, times variable, with LBRecovery, times variable, with LB Plating, incubation (37 C overnight)Plating, incubation (37 C overnight)

Bio-Rad:Bio-Rad:pGLO Bacterial pGLO Bacterial TransformationTransformation

Transform bacteria with jellyfish gene (GFP)Transform bacteria with jellyfish gene (GFP) Study gene selection and regulation (amp/ara)Study gene selection and regulation (amp/ara) Restriction enzyme and ligation conceptsRestriction enzyme and ligation concepts Advanced lab techniquesAdvanced lab techniques Possible extentions (GFP chromatography)Possible extentions (GFP chromatography) Complete in two 45 minute lab sessions Complete in two 45 minute lab sessions

Pros and ConsPros and Cons

+Highlights the central molecular +Highlights the central molecular framework of biology: framework of biology: (DNA(DNARNARNAProteinProteinTrait)Trait)

+Problem solving opportunities+Problem solving opportunities+/-Procedure is just complicated enough to +/-Procedure is just complicated enough to

have both and positive and negative have both and positive and negative outcomes (working v. not)outcomes (working v. not)

+Extention to GFP chromatography +Extention to GFP chromatography

Carolina Biological: E-Z Carolina Biological: E-Z Gene SplicerGene Splicer

Splice genes for ampicillin and kanamycin Splice genes for ampicillin and kanamycin resistance into a recombinant plasmidresistance into a recombinant plasmid

Transform E.Coli with the new plasmidTransform E.Coli with the new plasmid Isolate transformed bacteria by growing them Isolate transformed bacteria by growing them

on plates with ampicillin and kanamycinon plates with ampicillin and kanamycin Additional concept of ligation (gene slicing)Additional concept of ligation (gene slicing)

LigationLigation

Digested pAmp Digested pAmp w/ampicillinw/ampicillinresistance generesistance gene

++Digested pKANDigested pKANw/kanamycinw/kanamycinResistance geneResistance gene

++

Ligase w/ATP ---1 hour Ligase w/ATP ---1 hour plasmid w/ampicillin & kanamycin plasmid w/ampicillin & kanamycin resistance!resistance!

Carolina Pros and ConsCarolina Pros and Cons

+ All necessary supplies included+ All necessary supplies included

- Must streak plate bacteria ahead of time - Must streak plate bacteria ahead of time to obtain fresh colonies for transformation to obtain fresh colonies for transformation

Peyer Lab Systems: Cloning Peyer Lab Systems: Cloning a Fluorescent Gene a Fluorescent Gene (Granger!)(Granger!)

Amplification of GFP using PCRAmplification of GFP using PCR Ligation of GFP to a vector to make Ligation of GFP to a vector to make

recombinant plasmid for transformationrecombinant plasmid for transformation Transform E.Coli bacteria with the Transform E.Coli bacteria with the

recombinant plasmidrecombinant plasmid Isolate transformed bacteria by growing Isolate transformed bacteria by growing

on LB agar plate with ampicillinon LB agar plate with ampicillin Pix herePix here

PCRPCR

Peyer Pros and ConsPeyer Pros and Cons

+ Excellent lab manual+ Excellent lab manual

+ Can lease high cost equipment + Can lease high cost equipment (thermocycler, fluorescing light source, (thermocycler, fluorescing light source, micropipettes)micropipettes)

+ Pre-prepared agar plates + Pre-prepared agar plates

- PCR process and ligation must be - PCR process and ligation must be successful for lab to work effectivelysuccessful for lab to work effectively

Wards: Glowing Bacteria: Wards: Glowing Bacteria: Transformation with a Transformation with a Firefly GeneFirefly Gene

Introduction to biotechnology/plasmids Introduction to biotechnology/plasmids and their applicationsand their applications

Emphasizes precision in lab techniquesEmphasizes precision in lab techniques Bioluminescence using the Bioluminescence using the lucluc gene gene

Pros & ConsPros & Cons

+ interesting result if it works+ interesting result if it works + luciferin/luciferase relationship – discuss + luciferin/luciferase relationship – discuss

the action of enzymesthe action of enzymes - high cost- high cost - procedure has a lot of wait time; need - procedure has a lot of wait time; need

additional activity to fill this timeadditional activity to fill this time

Bio-Rad ProcedureBio-Rad Procedure

Pros: multiple labPros: multiple labtechniques, attention techniques, attention to procedural detail;to procedural detail;

highlights the central highlights the central molecular framework molecular framework of biology:of biology:(DNA(DNARNARNAProteinProteinTrait) Trait)