• Test efficiency of competent cells

Cell-Free Reactions

• Test cell-free extract reaction with T7-GFP plasmid

Reporter

• Transform T7 promoter, amilCP, and terminator
• Begin assembly by ligating linearized T7 promoter and amilCP

Reporter

• Transform T7-GFP plasmid, lacZ alpha fragment, and eGFP
• Send promising T7 promoter -- amilCP ligations to be sequenced
• Perform double digest of T7 promoter and terminator from last week
• Ligate double-digested T7 promoter to new reporters (lacZ and eGFP)

Cell-Free Reactions

• Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA

Toehold Switch

• Collins triggers activate the switches (both in plasmid form) to express LacZ

Reporter

• Ligate T7 promoter -- amilCP construct to terminator
• Extract successful ligations of T7 promoter to eGFP
• Ligate T7 promoter -- eGFP construct to terminator

Cell-Free Reactions

• Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA

Toehold Switch

• Collins plasmids express LacZ with 25 ng of switch
• DNA oligos trigger Collins switches

Reporter

• Identify successful ligations to terminator for amilCP and eGFP constructs using a gel
• Send correct plasmids for sequencing for confirmation
• Test plasmids in cell-free extract
• amilCP does not produce color in cell-free reaction
• eGFP produces fluorescence comparable to that from the Collins T7-GFP plasmid in cell-free reaction
• Linearized plasmids containing only the promoter and insert (no terminator) do not express protein

Cell-Free Reactions

• 384-well plate requires at least 10 μL of reaction

DNAzyme

• Anneal PO strand with catalytic strand, both with and without erbium
• Test success of annealing reaction in cell-free extract and with acrylamide gels

Reporter

• Sequenced amilCP construct does not contain amilCP
• Unsuccessfully linearize and amplify eGFP construct using PCR

Cell-Free Reactions

• Linear eGFP construct does not produce a stronger signal than its plasmid form

DNAzyme

• DNAzyme duplex does not trigger toehold switch
• Erbium cleaves the P substrate strand

Reporter

• Restart amilCP cloning process

Cell-Free Reactions

• Reactions can be diluted by one-half and still produce visible results in two hours

DNAzyme

• dPAGE assay of P substrate cleavage suggests that the DNAzyme works, but results are not definitive
• Six-hour time course of cleavage does not yield much additional information
• Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates

Reporter

• Continue amilCP cloning process

Amplifier

• Clone RBS-T3 RNA polymerase to add into other constructs

Cell-Free Reactions

• Reactions diluted by one-half produce significantly less protein than undiluted reactions

DNAzyme

• Annealing reactions produce hybrid complexes but leave unsequestered substrate strand
• DNAzyme does not cleave P substrate strand

Reporter

• Continue cloning amilCP construct
• Determine sequence of possible lacZ plasmid
• Unsuccessfully grow lacZ from iGEM bacterial stab

Amplifier

• Continue cloning T3 constructs

• Obtain fluorescence readings for UGA Archaeal InterLab

Cell-Free Reactions

• Presence of erbium and Buffer B as part of cleavage reaction does not seem to affect reaction progress

DNAzyme

• Higher catalytic-to-substrate strand ratios help increase sequestration of substrate strand, especially for G switch
• Cell-free reactions suggest that cleavage of the P substrate strand does occur in the presence of erbium
• dPAGE assay does not suggest cleavage

Reporter

• amilCP construct contains CFP, not amilCP
• Contact Collins lab for PT3-GFP construct

Amplifier

• Obtain T3 RNA polymerase gene via amplification
• Ligate PT3 and PT3-RBS into plasmid backbone

DNAzyme

• Begin work with lead DNAzyme
• dPAGE assay does not suggest cleavage occurs under the current conditions

Reporter

• Unsuccessful mutagensis of lacZ to remove EcoRI site for BioBricking

Amplifier

• Successfully ligate PT3-RBS into plasmid backbone
• Clone PT3-RBS-T3 and PT7-RBS-T3

• Complete data collection for InterLab study
• Prepare William & Mary plasmids for cell-free expression

DNAzyme

• Start working with hairpin lead DNAzyme
• Cell-free reactions suggest that a hairpin DNAzyme cleaves more efficiently than a DNAzyme in a duplex
• Adding the DNAzyme, substrate, and metal to a cell-free reaction produces greater switch activation
  than adding a completed cleavage reaction
• Cell-free reactions suggest that cleavage efficiency is similar at 37°C and room temperature

Reporter

• Unsuccessful mutagenesis of lacZ with DMSO

Amplifier

• Unsuccessfully ligate terminator onto PT3-T3 and PT7-T3 constructs

• Complete data collection for William & Mary (read our report here)

Amplifier

• ligate terminator onto PT3-T3 and PT7-T3 constructs

Cell-Free Reactions

• No reactions, including the positive control, turn purple to indicate the presence of LacZ

Reporter

• Unsuccessful mutagenesis of lacZ

• Obtain codon-optimized OFP, GFP, BFP, YFP, and RFP from 2015 Carnegie Mellon team

Cell-Free Reactions

• Plasmids from last week's reactions did not actually contain the toehold switch

DNAzyme

• Replicate results from Week 13 suggesting that hairpin lead DNAzyme sequesters and cleaves as expected

DNAzyme

• Presence of lead produces signal higher than background hairpin activation at 3.74 nM DNAzyme
• Presence of lead may produce signal beyond background activation at 50 nM and 100 nM DNAzyme

DNAzyme

• Presence of lead produces signal higher than background hairpin activation at 3.74 nM DNAzyme
• Presence of lead does not produce signal beyond background activation at 50 nM and 100 nM DNAzyme

Reporter

• Unsuccessful mutagenesis of lacZ

DNAzyme

• PAGE analysis suggests cleavage of hairpin but is not clear
• Presence of lead does not produce signal beyond background activation at 50 nM and 100 nM DNAzyme

Reporter

• Unsuccessful mutagenesis of lacZ

DNAzyme

• PAGE analysis suggests cleavage of hairpin but is not clear

Reporter

• Unsuccessful mutagenesis of lacZ

• Characterize codon-optimized fluorescent proteins from 2015 Carnegie Mellon team

Cell-Free Reactions

• Expected dosage response to lead not observed
• Expected lack of activation with D switch not observed
• Reaction colors do not look the same as in previous weeks

Reporter

• Unsuccessful mutagenesis of lacZ

• Finish cloning Parts
• Submit Parts