Team:Pittsburgh/Notebook
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Our weekly activities: experiments, data analysis, and planning. For a list of our protocols, visit the Protocols page.
Contents
- Week 1: May 23 - May 27
- Week 2: May 31 - June 3
- Week 3: June 6 - June 12
- Week 4: June 13 - June 17
- Week 5: June 20 - June 26
- Week 6: June 27 - July 3
- Week 7: July 5 - July 8
- Week 8: July 11 - July 17
- Week 9: July 18 - July 22
- Week 10: July 25 - July 31
- Week 11: August 1 - August 5
- Week 12: August 8 - August 13
- Week 13: August 15 - August 20
- Week 14: August 23 - August 26
- Week 15: August 29 - September 2
- Week 16: September 6 - September 9
- Week 17: September 12 - September 17
- Week 18: September 19 - September 24
- Week 19: September 28 - September 30
- Week 20: October 3 - October 8
- Week 21: October 10 - October 14
- Week 22: October 17 - October 19
- Week 23: October 24 - October 31
Week 1: May 23 - May 27
- Training begins
- Grow Top 10 competent cells.
- Brainstorm genetic circuits for a thallium sensor
- Lab safety training
Week 2: May 31 - June 3
- Test efficiency of competent cells
Cell-Free Reactions
- Test cell-free extract reaction with T7-GFP plasmid
- Contact museums and summer programs for outreach opportunities
- Lab safety training
Week 3: June 6 - June 12
Reporter
- Contact museums and summer programs for outreach opportunities
Week 4: June 13 - June 17
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)
- TECBio, DiSCoBio, and Camp BioE outreach opportunities set
Week 5: June 20 - June 26
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
- Reach out to teams to collaborate based on last year's projects
Week 6: June 27 - July 3
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
- Work on outreach presentation for Camp BioE
Week 7: July 5 - July 8
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
- Practice outreach presentation for Camp BioE
- Develop DNAzymes for other heavy metals
Week 8: July 11 - July 17
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
- First presentation at Camp BioE
- Prepare for UMD Mid-Atlantic Meet-Up
- Contact PLSG and NEB for sponsorship
Week 9: July 18 - July 22
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
- Presentation at Camp BioEhttps://2016.igem.org/Team:Pittsburgh/Collaborations#UMD
- UMD Mid-Atlantic Meet-Up
- Continue fundraising
- Discuss systems to model
Week 10: July 25 - July 31
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
- Presentation at Camp BioE
- Start modeling toehold kinetics and the economical effects of lead
Week 11: August 1 - August 5
- 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
- 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
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
- Table at Carnegie Science Center's H2Oh! Exhibit
- Work on Experiment.com video for crowdfunding
Week 13: August 15 - August 20
- 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
- Work on Experiment.com video for crowdfunding
Week 14: August 23 - August 26
- Complete data collection for William & Mary (read our report here)
Amplifier
- ligate terminator onto PT3-T3 and PT7-T3 constructs
- Meet with Dr. Troesken from Pitt's Department of Economics to discuss lead population model
Week 15: August 29 - September 2
Cell-Free Reactions
- No reactions, including the positive control, turn purple to indicate the presence of LacZ
Reporter
- Unsuccessful mutagenesis of lacZ
- Move lab up to campus
Week 16: September 6 - September 9
- 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
- Experiment.com campaign approved
Week 17: September 12 - September 17
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
- Experiment.com campaign is live
Week 18: September 19 - September 24
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
- Modeling
Week 19: September 28 - September 30
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
- Modeling
Week 20: October 3 - October 8
DNAzyme
- PAGE analysis suggests cleavage of hairpin but is not clear
Reporter
- Unsuccessful mutagenesis of lacZ
Week 21: October 10 - October 14
- 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
- Modeling
- Finalize Wiki
Week 22: October 17 - October 19
- Finish cloning Parts
- Submit Parts
- Finalize Wiki
Week 23: October 24 - October 31
- Prepare poster and presentation
- Giant Jamboree!
