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

Wet Lab

• Test efficiency of competent cells

Cell-Free Extract

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

Dry Lab

• Contact museums and summer programs for outreach opportunities
• Lab safety training

Week 2 Notebook
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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 3 Notebook
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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 Tissue Engineering Camp outreach opportunities set

Week 4 Notebook
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Week 5: June 20 - June 26

Wet Lab

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

• 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 5 Notebook
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Week 6: June 27 - July 3

Wet Lab

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 consturcts 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

• Work on outreach presentation for tissue engineering camp

Week 6 Notebook
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Week 7: July 5 - July 8

Wet Lab

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

Dry Lab

• Practice outreach presentation for tissue engineering camp
• Develop DNAzymes for other heavy metals

Week 7 Notebook
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Week 8: July 11 - July 17

Wet Lab

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

Dry Lab

• First presentation at Camp BioE
• Prepare for UMD Mid-Atlantic Meet-Up
• Contact PLSG and NEB for sponsorship

Week 8 Notebook
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Week 9: July 18 - July 22

Wet Lab

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

Dry Lab

• Presentation at Camp BioE
• UMD Mid-Atlantic Meet-Up
• Continue fundraising
• Discuss systems to model

Week 9 Notebook
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