IGEM:MIT/2005/Friday 8/5 Meeting Agenda and Minutes

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Contents

General Updates

  • Monday meeting TBRun by Maxine
  • Assemblies: at beginning of week, we're planning on doing plating -> ON -> miniprep of all constructs needed for upcoming assemblies.
    • This turns a ~4 day process into a ~2 day process
  • Updated flowchart?
  • Registry problems seem to be worked out...
    • Should make glycerols for registry of all parts made or assembled
  • Will start transitioning to Endy lab early next week, probably.
  • Look at all the green on our parts site! Wow!

SubTeam Updates

Format: what is the progress? what are the issues?

Input: Maxine

1. Fluorescein and Oligo Uptake by competent (lots of competent cells made if anyone needs them) and overnighted MC4100 culture

0-no fluorescence
5-high fluorescence

Competent cells, fluorescein: 5
Competent cells, m9: 2.5
Competent cells, oligo: 1

Overnight cells, fluorescein: 1
Overnight cells, m9: 1
Overnight cells, oligo: 2

Should I post pics online?

2. Effects of Various Fluorescein Concentrations on MC4100 Overnight Culture Experiment

a) FACS: Fluorescence increased w/increasing conc for the most part. Should I get help for more detailed analysis?
b) plating results:

control: 5.2 X 10^8 cells/ml
5M: 4.5 X 10^8 cells/ml
500mM: 4.7 X 10^8 cells/ml
50mM: 1.60 X 10^9 cells/ml
5mM: 7.3 X 10^8 cells/ml
500microM: 1.26 X 10^9 cells/ml
50microM: 1.03 X 10^9 cells/ml
5microM: 3.2 X 10^8 cells/ml

Receiver Head-Unit: Jenny

Progress: scFv Testing
Image:Check.gif Materials (most?) for western blot.
Image:Check.gif Promoter.RBS biobrick BBa_J13002.
Image:Pending.gif Primer start-codon for scFv
Progress: ToxR Unit
Image:Check.gif scFv.TT Biobricked. BBa_J07043, 44, 45.

Receiver/Transmitter 1 - ToxR : Wiki-Will

Progress:

  • assembling ctx-promotor (J07007) :: gfp composite.
    • transform failed --> plate showed no growth this morning.
    • i'd like to think we'll get this piece working this weekend.

Problems:

  • waiting on ToxR (drew, how about your "i can assemble it in a week, wait forget i said that." i don't want to forget.)


*want to review the system?
http://openwetware.org/index.php?title=Image:SystemToxRpathway.jpg
Image:Check.gif scFv, with terminator
Image:Check.gif malE , negative control
Image:Pending.gif PhoA, positive control. Ready, but still must remove restriction site with quikchange (Hi Annie!)
Image:Pending.gif fusion of scFv, malE, and PhoA to ToxR. (for obvious reasons)
_
Image:Check.gif have constitutive promotor :: rbs part
Image:Pending.gif fusion to ToxR ...
_
Image:Pending.gif ctx promotor :: gfp ***almost done***

Receiver/Transmitter 2 - FecA : Annie

  • Progress:
-Worked out details of all building processes Annie's Notes
-Specified more parts J07046-J07063 composite parts for FecA system 
-Base parts made: FecA, FecI, FecR, attempted FecA'
-Base parts plan to make next week: FecA', FecR', FecA Promoter, 
-Assembling: rbs with FecI and FecR, rbs with promoter 
-Have overnight culture and a plate of DY329, ready for making FecA- and Fur-
  • Problems:
-2 of 5 primers for adding linker to scFv are wrong. Add a different aa sequence
 5'RVARAVTTRSRISTRSRSG 3' instead of 5'PNSASHSGSAPNTSSAPGS 3'
-Primers for fusion are wrong. If we go with the accidentally-created linker, we can use half
 of the already ordered primers --> reorder the other half.  
  • Solutions:
-reorder

Signal Processor: Ray

  • Waiting on parts from registry to do own assembly

Actuator

  • Have begun using E0840 (rbs:gfp:TT) in conjunction with other systems


Issues/Discussion

  1. Input: having trouble with repeatable results
  2. Do we know that our input molecules are what we think they are?
  3. To look at experiment reproducibility, be consistent!
  4. Still waiting for ToxR -- Can I call?
  5. Sequencing: get info from biopolymer -- need plasmid DNA and primers
    • Everything we've assembled, get sequenced!!
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