1. iGEM Bioware
Summer Week 7!

2. Overview
Arsenic
Gold
Decoder
Lock & Key
Other

3. Arsenic Collection System
Most PCRs didn't work (10% success rate!), and we're waiting on primers and restriction enzymes. The PCRs that worked showed ArsR and pArsR:
Also we digested Amp backbone so we are ready to ligate in ArsR.
Transformed pArsR into pSB1T3 this AM.
1kb                pArsR          100bp
                with primers
                  F-255, R-2
1kb   ArsR!
3kb
2kb
1.5kb
1kb
.5kb

4. Gel of pSB1T3 and pArsR digestions
We did a gel extract, ligation, 
and then transformed cells this morning.
Undigested/Single Digest
Vector Backbone
Insert (RFP)
3kb
2kb
1.5kb
1kb
.5kb
pArsR

5. Gold Collection System
GolB - digested, ligated into pSBIA2
GolS - digested, ligated into pSBIA2
GesABC - ?
GolT - ?

6. The gel on right shows bands only in lanes with no template.
Recent attempt to clone out GesABC and GolT with GolB and GolS as positive controls
The gel on right shows bands only in lanes with no template.
GolT -T
GolB -T
GolS -T

7. Gold Collection System
Still trying to clone out GesABC and GolT
Ran PCR program with elongated extension time (12 min)
   degC for 2 min
   degC for 30 sec
   degC for 30 sec
   degC for 12 min
   5. Repeat from step 2 for 30 cycles
   6. Final 68 degC for 10 min
GesABC was a 50 ul reaction
    2 ul PrimerF, 2 ul PrimerR, 2 ul polymerase, 2 ul dNTP, 16 ul template, 5 ul buffer, 21 ul H2O

8.  ( gel)

9. Digestion, Gel extraction
*full digest with XbaI and SpeI unless otherwise indicated
*blue boxes show which bands we cut out

10. Yesterday, we ran gradient PCR with different annealing temperatures
Tried 48, 50, and 52 degC for GesABC and GolT
         51, 53, and 55 degC for GolB and GolS

11. Decoder sRNA target biobricks have put into pSB1AC3F (RFC25). OmpF
OmpA
GadX
New Primers have been ordered to fit sRNA target biobricks into pSB1A3 (RFC 10).  
sRNA biobricks have been put into pSB1T3
MicF
MicA
GadY
These regulators will have 8 extra nucleotides in their 5' end (biobrick scar)
When will we have the computer back for the plate reader?

12. Lock and Key Decoder   
Spent the majority of this week waiting on restriction enzymes.  

13. Questions Other Updates Plate reader?
Is there a way to prevent the gel from being half dark?
Nano-drop and PCR purification consistency?
Other Updates
Haven't heard back from Midsci, but Brian Gallagher from GE Healthcare called about donating products.
Volleyball tournament postponed until just after school starts.