Course Project Engineering electricity production by plants http://biophotovoltaics.wordpress.com/

Game plan Engineering electricity production by plants 1. Learn more about how “plants” harvest energy and how to improve it

Game plan Engineering electricity production by plants Learn more about how “plants” harvest energy and how to improve it Learn more about how to transform light energy into electricity by way of photosynthesis

Game plan Engineering electricity production by plants Learn more about how “plants” harvest energy and how to improve it Learn more about how to transform light energy into electricity by way of photosynthesis Pick some organisms (or groups of organisms) to study

Game plan Engineering electricity production by plants Learn more about how “plants” harvest energy and how to improve it Learn more about how to transform light energy into electricity by way of photosynthesis Pick some organisms (or groups of organisms) to study Design some experiments

Game plan Engineering electricity production by plants Learn more about how “plants” harvest energy and how to improve it Learn more about how to transform light energy into electricity by way of photosynthesis Pick some organisms (or groups of organisms) to study Design some experiments See where they lead us

Game plan Engineering electricity production by plants Learn more about how “plants” harvest energy and how to improve it Learn more about how to transform light energy into electricity by way of photosynthesis Pick some organisms (or groups of organisms) to study Design some experiments See where they lead us

Grading? Combination of papers, presentations & lab reports 4 lab reports @ 2.5 points each 5 assignments @ 2 points each Presentation related to project: 5 points Research proposal: 10 points Final presentation: 15 points Poster: 10 points Draft report 10 points Final report: 30 points Assignment 1 Pick a photosynthetic organism that might be worth studying Try to convince the group in 5-10 minutes why yours is best: i.e., what is known/what isn’t known

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well Add restriction sites at 5’ end: may need to reamplify an amplicon

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well Add restriction sites at 5’ end: may need to reamplify an amplicon Use Vent or another polymerase with proof-reading , taq’s error frequency is too high.

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations Amplifying sequences from related organisms If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations Amplifying sequences from related organisms If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities CodeHOP is a way around this: have a perfect match for 10-12 bases at 3’ end, then pick most likely candidates for the rest.

Optimizing PCR Choosing enzyme Template (RNA or DNA?)

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation)

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation) Fragment ends

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation) Fragment ends Cost

Optimizing PCR Choosing enzyme Template (RNA or DNA?): Reverse transcriptases from retroviruses make DNA copies of RNA Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn2+

Optimizing PCR Choosing enzyme Template (RNA or DNA?): Reverse transcriptases from retroviruses make DNA copies of RNA Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn2+ Then dilute rxn & add Mg2+ to do PCR

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn2+ Then dilute rxn & add Mg 2+ to do PCR Tfl DNA Polymerase from Thermus flavus has no RT activity: can mix with RNA & RT w/o activity then go directly to PCR after RT is done

Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading

Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000

Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000 Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000

Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000 Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000 Genetically engineered proof-reading Phusion from NEB has error frequency of 1 in 2,000,000

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C T1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C T1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C T1/2 of Vent @ 95˚ C is 6.7 hours, 1.8 hours @ 100˚ C

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C T1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C T1/2 of Vent @ 95˚ C is 6.7 hours, 1.8 hours @ 100˚ C T1/2 of Deep Vent from Pyrococcus species GB-D (grows @ 104˚ C)is 23 hours @ 95˚ C, 8 hours @ 100˚ C

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Phusion is 10x more processive than Pfu, 2x more than Taq

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Phusion is 10x more processive than Pfu, 2x more than Taq lets you make longer amplicons in shorter time

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Phusion is 10x more processive than Pfu, 2x more than Taq lets you make longer amplicons in shorter time Taq = 8 kb max cf 40 kb for Phusion

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km dNTP: 13 µM for Taq, 60 µM for Vent

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km dNTP: 13 µM for Taq, 60 µM for Vent DNA: 2 nM for Taq, 0.01 nM for Deep Vent

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax: >1,000 nt/s when attached Binding is limiting, processivity determines actual rate 1000 bp/min is good for PCR

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion dNTP analogs or modified dNTP non-proofreading polymerases do better, but varies according to the modification

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion dNTP analogs or modified dNTP non-proofreading polymerases do better, but varies according to the modification [Mg]: Vent is more sensitive to [Mg] and needs 2x more than Taq

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Non-proof-readers (eg Taq) give a mix of blunt & 3’A

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Non-proof-readers (eg Taq) give a mix of blunt & 3’A Can use 3’A for t:A cloning GAATTCAtcgca CTTAAGtagcgt

Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Cost @ NEB: http://www.neb.com/nebecomm/default.asp Taq = $59.00 for 400 units Vent = $62.00 for 200 units Deep Vent = $90.00 for 200 units Phusion = $ 103.00 for 100 units

Optimizing PCR [enzyme] [Template] [Mg2+] Annealing Temperature Denaturation temperature

Optimizing PCR [enzyme] 0.4-2 units/100 µl for proofreaders : start with 1

Optimizing PCR [enzyme] 0.4-2 units/100 µl for proofreaders : start with 1 1-5 units/100 µl for non-proofreaders : start with 3

Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids 10 - 1000 ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants

Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids 10 - 1000 ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants [dNTP] 50-500 µM for Taq: start with 200, lower increases fidelity, higher increases yield 200-400 µM for proof-readers: if too low start eating

Optimizing PCR [enzyme] [Template] [Mg2+] 0.5 - 4 mM for Taq: start with 1.5; lower if extra bands, raise if low yield

Optimizing PCR [enzyme] [Template] [Mg2+] 0.5 - 4 mM for Taq: start with 1.5; lower if extra bands, raise if low yield 1- 8 mM for proofreaders: start with 2, lower if extra bands, raise if low yield

Optimizing PCR [enzyme] [Template] [Mg2+] Denaturation Temperature Go as high as you can w/o killing enzyme before end 94˚C for Taq 96-98˚C for Vent 98˚C for Deep Vent & Phusion

Optimizing PCR [enzyme] [Template] [Mg2+] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm

Optimizing PCR [enzyme] [Template] [Mg2+] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed

Optimizing PCR [enzyme] [Template] [Mg2+] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed # cycles: raise if no bands, lower if OK yield but extra bands

Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles

Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles Can try “PCR enhancers” to overcome dirty DNA Use Ammonium SO4 in buffer cf KCl Use molecules that alter Tm eg DMSO & formamide Use molecules that stabilise Taq eg Betaine & BSA

Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles If extra bands persist, use Taq bound to antibody Inactive until denature antibody 7’ at 94˚ C

Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles If extra bands persist, use Taq bound to antibody Inactive until denature antibody 7’ at 94˚ C Alternatively, try touch-down: start annealing @ too high & lower 1˚ C each cycle ( binds correct target first)