IGEM:MIT/2005/Friday 8/26 Meeting Agenda and Minutes

General Updates

• Who's leaving? Who's coming back? When

Jen - leaving today - back when school starts

Maxine - leaving Tuesday

Annie - leaving Tuesday

Will - ?

Ray - left due to illness

Jessica - back on Monday

• Semester meeting schedule - Let's try Friday at 5pm. The meetings will necessarily be short. We can always try something else.

Subteam Updates

Input-Maxine

• Doing experiment on microscope today
• Is there any access to any other fluorescent microscope? I waited a few days to use it.

Head Unit-Jen

• Backtracked a few weeks, decided to not just use the PCR product, but instead put it into vector
• Made new Biobricks (need to be specified) consisting of ATG-scFv (x3)
• To do: assembly with promoter::RBS construct, western blot testing.
• Does someone want to take over? If so we need to talk about it today.

FecA-Annie

• Construction of FecA Pro-RFP, FecA Pro-GFP

Failed once

Did again and got lots of colonies for Feca Pro-GFP, but 2 - controls got some colonies as well.

-Having Registry assembling as well

• PhoA

2nd QuikChange no colonies

Problems:

Optimized version of PhoA in Registry, 81% similarity by BLAST

PhoA Registry has 1 PstI & 1 EcoRI site. PhoA NCBI has 2 EcoRI sites. Diagnosis digestion confirmed this.

All primers designed for PhoA (PCR & restriction sites removal) were based on PhoA Registry

Solution:

Reorder primers to PCR and remove restriction sites to get the "real" PhoA

• FecA

3rd QuikChange resulted in no colonies

Doing 3-step PCR (running gel to confirm)

• scFv-Linker

confirming whether or not Linker was added

ordered primers for sequencing

Actuator-Jessica

• ToxR system:

ctx assembled w/ GFP

ctx assembling w/ RFP

• FecA system: parallel

FecA Promoter assembling w/ RFP

FecA Promoter assembling w/ GFP