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Bioware Lab Work: Bootcamp on Constructing Biobricks and Fluorescent Protein Analysis

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Join our Bioware Lab Bootcamp where we focus on cloning and combining Biobricks from our parts registry to create plasmids. Projects include using constitutive and UV-inducible promoters with ECFP, YFP, and GFP fluorescent proteins. Participants will learn essential techniques such as plasmid transformation, miniprep, digestion, ligation, and selection of non-RFP cells. The course culminates in fluorescence reading and PCR confirmation of Biobrick sequences. Ideal for those interested in synthetic biology and genetic engineering.

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Bioware Lab Work: Bootcamp on Constructing Biobricks and Fluorescent Protein Analysis

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  1. Bioware Lab Work – Bootcamp! Clone two Biobricks from the parts registry and combine them in a plasmid.

  2. Projects • Tom and Amanda – constitutive pTet promoter and ECFP • Francis and Bob – • Steve and Matt – UV inducible promoter and YFP • Meagan and Erin – constitutive pTet promoter and GFP (parts E0240 and E0840) Plasmid Backbone from J04450: CAP resistance and RFP Biobrick

  3. Procedure • Remove plasmids from registry – Transform • Miniprep, Digest, Ligate, Transform • Select for non RFP cells that grow on CAP • 2 overnight cultures to prepare for a fluorescence reading • Confirmation using PCR amplification of Biobrick sequequence inside the prefix and suffix.

  4. Plates Meagan and Erin – 12M | 120 pTet + GFP Transformations 5/26/2010 RFP Tom and Amanda – pTet + ECFP Transformations 5/26/2010

  5. Promoter Digest 2% Gel 1 2 3 4 5 6 7 8 9 10 1. 1 kb ladder 2. Constitutive promoter 3. UV promoter 4. Rx1 Promoter 5. 18C promoter 1 6. 18 C promoter 2 7. RFP 8. Empty 9. Empty 10. Empty

  6. Digestions 1% Gel 1. 1 kb ladder 2. ECFP 3. RFP (for backbone) 4. YFP 5. RFP (for backbone 6. Downstream Part? 7. RFP (for backbone) 8. 12O GFP 9. 12M GFP 10. RFP (for backbone) 1 2 3 4 5 6 7 8 9 10

  7. Fluorescence Picture

  8. Possible Directions • Super metal-resistant cell • Magnetosomes • Metal mobilization • Metal immobilization

  9. Metal Resistant Cell

  10. Metal Resistant Cell

  11. Past Projects • 2009 • Cornell-Cd sensor-bronze • Groningen-As accumulation/remediator-gold, finalist • Seoul-Cd As Zn sensor • Newcastle-Cd sensor/remediator-gold • Tokyo Tech-Fe oxidation-silver • Australia-Hg accumulation, ice precipitation-bronze • Virginia-As accumulation-bronze • 2008 • UNAM Mexico-Ni Transport-bronze • METU-Turkey-heavy metal • sequestration • Prairieview-Cation transporters-bronze • 2007 • St. Petersburg-Cu sensor • Brown-Pb sensor • MIT-Hg sensor/deactivator • Prairieview-Fe(II), V(II), Ni(II), S sensor • Turkey-Metal Transporter • 2006 • Edinburgh-As sensor • Latin America-Fe sensor

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