hisGFPmek and PDZ domainHis6 tag

(purification)Mek2 tag(PDZ binding)

*not to scale, at all

G H H

H H

H H

G

Q P G T P T R T A V

hisGFPmek protein

PDZ domain

E. coli

LppOmpA

Initial cell binding assay

PBS/BSA JsLO, no GFP JsLO + hisGFPmek JsLOPDZ1 + hisGFPmek JsLOPDZ2 + hisGFPmek0

50

100

150

200

250

300

350

400

450

500

Ex 488, Em 507(published forEGFP clone)

In vitro hisGFPmek bindingassay with GST-PDZ fusion protein

--GSH

Glutathione bead

GST(glutathione S-transferase)

PDZ

Q P

G T

P T

R T

A V

hisGFPmek

PBST

GST-PDZ'd

GSH bea

ds

hisGFP

mek on GSH

beads

hisGFP

mek on GST

-PDZ'd bea

ds0

20

40

60

80

100

120

140

160

180

In vitro hisGFPmek assay

Ex 485, Em 538(quorum-sensinggroup protocol)

0

200

400

600

800

1000

1200

1400

In vitro hisGFPmek assay

Ex 488, Em 507(published forEGFP clone)

Rebuilding the Voigt construct

Voigt et al., 2006

• Amplification by PCR, digestion/ligation of PCR’d fragments • Pros

– Faster, more processive protocol– No overnight liquid cultures or transformations (until the end)– No gel isolation, just PCR purification– No colony PCR– PCR is better amplifier than bacteria– Can reconstitute unavailable subparts

• Cons– Can’t save intermediate constructs (except by cloning PCR products

into Topo vector)– Requires primer design and synthesis– Problems with identical/homologous BioBrick parts

Alternative PCR/digest assembly

Alternative PCR/digest assembly

BBa_J23039BBa_T9002

E X S P X S P VRE

BB_F

BBa_T9002

P VRE

VRE

SX

PSX

BB_R as well as BB_F

P VRE SX

SE X P

BB_R

Plans

• Explore AIDA construct– AIDA-streptavidin– AIDA-PDZ

• Make a new hisGFPmek?– Longer Mek tag, or longer linker

• Finish the Voigt construct by PCR/digest– Forever abandon traditional BioBricks protocol?

Alternative PCR/digest assembly

BBa_T9002

P VRE

VRE

SX

PSX