IGEM:IMPERIAL/2009/4Sep/System Overview/Mod1

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Contents

Module 1

Module function in overall design

  • High level description:

This phase is for protein production. Our two proteins of interest are

1) PAH

2) Cellulase

Production is initially repressed by LacI. When IPTG is added, derepression occurs, protein production starts.


  • Input(s)/Output(s):

Input: IPTG.

Output: Protein of interest.

HIGH IPTG = Production of protein LOW IPTG = Protein production repressed

Assays to be done

Assays Importance (1=Most important. 3=Least important) When? (1=Now. 3=Last) Variable Rationale Output Model
Influence of IPTG on cell growth 2 2 IPTG Influence of IPTG on protein production & cell growth IPTG Yes
Cellulase (activity) 2 3 - Data analysis Activity MM
PAH (activity) 2 3 - Data analysis Activity MM
Cellulase concentration 3 3 - Data for modelling Activity MM
PAH concentration 3 3 - Data for modelling Activity MM


Data we hope to Get from this

  • IPTG cell growth assay

Curve telling us optimum amounts of IPTG for maximum cell growth.

  • How essential is this data for the success of the project (0..10)

5

Testing Construct Design

  • How many of them ?

2 of them. 1 for PAH, 1 for cellulase

  • Picture/Diagram for all test constructs

Image:II09_M1_C3.jpg
Image:II09_TC_C4.jpg
Image:II09_M1_C5.jpg
Image:II09_M1_C6.jpg
Image:II09_M1_C7.jpg
Image:II09_M1_C8.jpg
Image:II09_M1_C9.jpg
Image:II09_M1_C10.jpg
Image:II09_M1_C11.jpg


  • Describe briefly purpose behind each test construct

For both cellulase and PAH, RFP fluorescence is the output. This allows us to determine if cellulase and PAH are being produced from respective constructs.

Progress

  • Current status in terms of cloning for each assembly

Image:II09_WL TC Key.jpg Image:II09_WL TC M1.jpg


  • Number of cloning steps remaining before testing construct


  • Gene Synthesis involved ? (if yes, when is it due ?)

Yes. Due date up to gene art, probably by next week.

  • How likely are we to get the cloning finished before [Jamboree - 2 weeks] (deadline to send parts) ? [unlikely, likely, certain]

Likely

Data we hope to Get from this

  • Fore each test construct, sketch graph or write description, but make sure it is clear and understanable

For both cellulase and PAH, we hope test the activity of protein of interest produced, for dosage control calculations. Activity can be obtained from calibration curves obtained via the assays.

  • How essential is this data for the success of the project (0..10)

9. We need the production of protein of interest.

Ready for testing construct

  • Protocols written and reviewed ?

1) PAH - Protocol from sigma. Reagents to be bought.

2) Cellulase - test kit to be bought

  • Any expertise needed? Acces to specific equipment needed ?

1) PAH - Spectrophotometry @ 450nm

2) Cellulase - Fluorescence microplate reader at excitation/emission settings of 360/460 nm

  • Modelling done ? Ready to analyse experimental data ?

Yes, Link to M1 modelling page and yes.

Comments

  • Additional info you think is important.
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