Bioware Lab Work – Bootcamp!

Clone two Biobricks from the parts registry and combine them in a

plasmid.

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

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.

Plates

Meagan and Erin – 12M | 120pTet + GFP Transformations5/26/2010

Tom and Amanda – pTet + ECFP Transformations5/26/2010

RFP

Promoter Digest 2% Gel 1 2 3 4 5 6 7 8 9 10 1. 1 kb ladder

2. Constitutive promoter3. UV promoter4. Rx1 Promoter 5. 18C promoter 16. 18 C promoter 27. RFP8. Empty9. Empty10. Empty

Digestions 1% Gel 1 2 3 4 5 6 7 8 9 10 1. 1 kb ladder

2. ECFP3. RFP (for backbone)4. YFP5. RFP (for backbone6. Downstream Part?7. RFP (for backbone)8. 12O GFP9. 12M GFP10. RFP (for backbone)

Fluorescence PictureGFP YFP RFP GFP

Exitation 501 512 584 439

Emission 511 529 687 476

Possible Directions

• Super metal-resistant cell• Magnetosomes• Metal mobilization• Metal immobilization

Metal Resistant Cell

Metal Resistant Cell

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