Team:Pittsburgh/Notebook
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Our weekly progress. For a list of our protocols, visit the Protocols page
Contents
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 Extract
- 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 Extract
- 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 Extract
- 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 Extract
- 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 Extract
- 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 Extract
- 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 Extract
- 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
DNAzyme
Reporter
Amplifier
- Presentation at Camp BioE
- Meeting with Dr. Daniel Bain from the University of Pittsburgh Department of Geology and Environmental Science
