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Revision as of 17:51, 18 October 2016

Our weekly activities: experiments, data analysis, and planning. For a list of our protocols, visit the Protocols page.

Week 1: May 23 - May 27

Wet Lab Dry Lab

Week 2: May 31 - June 3

Wet Lab

Cell-Free Reactions

Dry Lab

Week 3: June 6 - June 12

Wet Lab

Reporter

  • Transform T7 promoter, amilCP, and terminator
  • Begin assembly by ligating linearized T7 promoter and amilCP
Dry Lab
  • Contact museums and summer programs for outreach opportunities

Week 4: June 13 - June 17

Wet Lab

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)
Dry Lab
  • TECBio, DiSCoBio, and Camp BioE outreach opportunities set

Week 5: June 20 - June 26

Wet Lab

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
Dry Lab
  • Reach out to teams to collaborate based on last year's projects

Week 6: June 27 - July 3

Wet Lab

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
Dry Lab

Week 7: July 5 - July 8

Wet Lab

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
Dry Lab
  • Practice outreach presentation for Camp BioE
  • Develop DNAzymes for other heavy metals

Week 8: July 11 - July 17

Wet Lab

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
Dry Lab

Week 9: July 18 - July 22

Wet Lab

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
Dry Lab

Week 10: July 25 - July 31

Wet Lab

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
Dry Lab
  • Presentation at Camp BioE
  • Start modeling toehold kinetics and the economical effects of lead

Week 11: August 1 - August 5

Wet Lab

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
Dry Lab
  • Presentation at Camp BioE
  • Meeting with Dr. Daniel Bain from the University of Pittsburgh Department of Geology and Environmental Science
  • Model population effects of lead without economic layer

Week 12: August 8 - August 13

Wet Lab

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
Dry Lab

Week 13: August 15 - August 20

Wet Lab
  • 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
Dry Lab
  • Work on Experiment.com video for crowdfunding

Week 14: August 23 - August 26

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

Amplifier

  • ligate terminator onto PT3-T3 and PT7-T3 constructs
Dry Lab
  • Meet with Dr. Troesken from Pitt's Department of Economics to discuss lead population model

Week 15: August 29 - September 2

Wet Lab

Cell-Free Reactions

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

Reporter

  • Unsuccessful mutagenesis of lacZ
Dry Lab
  • Move lab up to campus

Week 16: September 6 - September 9

Wet Lab
  • 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
Dry Lab
  • Experiment.com campaign approved

Week 17: September 12 - September 17

Wet Lab

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
Dry Lab
  • Experiment.com campaign is live

Week 18: September 19 - September 24

Wet Lab

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
Dry Lab
  • Modeling

Week 19: September 28 - September 30

Wet Lab

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
Dry Lab
  • Modeling

Week 20: October 3 - October 8

Wet Lab

DNAzyme

  • PAGE analysis suggests cleavage of hairpin but is not clear

Reporter

  • Unsuccessful mutagenesis of lacZ

Week 21: October 10 - October 14

Wet Lab
  • 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
Dry Lab
  • Modeling
  • Finalize Wiki

Week 22: October 17 - October 19

Wet Lab
  • Finish cloning Parts
Dry Lab
  • Finalize Wiki