iGEM Bioware

Summer Week 7!

Overview

ArsenicGoldDecoderLock & KeyOther

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   ArsR!

1kb                pArsR          100bp                with primers                  F-255, R-2

3kb2kb1.5kb1kb

.5kb

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)

pArsR

3kb2kb1.5kb1kb

.5kb

Gold Collection System

GolB - digested, ligated into pSBIA2GolS - digested, ligated into pSBIA2GesABC - ?GolT - ?

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

GolB-T

GolT-T

GolS-T

Gold Collection System

Still trying to clone out GesABC and GolTRan PCR program with elongated extension time (12 min)   1. Denature @ 92 degC for 2 min   2. Denature @ 92 degC for 30 sec   3. Annealing @ 51 degC for 30 sec   4. Extension @ 68 degC for 12 min   5. Repeat from step 2 for 30 cycles   6. Final Extension @ 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

 (7.8.10 gel)

Digestion, Gel extraction*full digest with XbaI and SpeI unless otherwise indicated

*blue boxes show which bands we cut out

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

DecodersRNA target biobricks have put into pSB1AC3F (RFC25).OmpFOmpAGadXNew Primers have been ordered to fit sRNA target biobricks into pSB1A3 (RFC 10).  

sRNA biobricks have been put into pSB1T3MicFMicAGadYThese regulators will have 8 extra nucleotides in their 5' end (biobrick scar)

When will we have the computer back for the plate reader?

Lock and Key Decoder   

Spent the majority of this week waiting on restriction enzymes.  

QuestionsPlate 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.