IGEM:IMPERIAL/2009/4Sep/System Overview/Mod2

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Contents

Module 2

Module function in overall design

  • High level description:

The encapsulation phase is induced by the utilisation of the secondary carbon source and is designed to protect the chassis against dessication and acid exposure.


  • Induction is controlled by the PcstA promoter.
  • Dessication is protected against via the biosynthesis of the disaccharide trehalose.
  • Acid exposure is protected against by two factors:

1) A physical barrier of negativly charged colanic acid. The coat mops up hydrogen ions slowing their entry into the cell.

2) The upregulation of numerous membrane-stress proteins.

Assays to be done

Assays Importance (1=Most important. 3=Least important) When? (1=Now. 3=Last) Variable Rationale Output Model
CRP response to varying glucosetd> 2 1 Glucose Influence of glucose on CRP promoter output Fluorescence In progress
CRP and secondary carbon source 3 - 2nd carbon source Varying output with different carbon sources Fluorescence In progress
Qualitative visual analysis of colanic acid production 1 2 Construct Different phenotypes with different constructs Visual N/A
Quantitative: Quality measurement colanic acid 1 2 Construct "Different constructs will give varying levels of protection Amount & efficiency of colanic acid coat." OD, PCV, Fluorescence N/A
Trehalose 2 - N/A Detection of trehalose Fluorescence N/A



  • Current status in terms of cloning for each assembly:

Image:II09_WL TC Key.jpg Image:II09_WL TC M2.jpg



M2 Induction

  • Input(s)/Output(s):

The PcstA assay will investigate the transition between glucose and the secondary carbon source on the PcstA promoter. The output from the promoter is measured by fluorescence.

Testing Construct Design

  • How many of them?:

There is one testing construct for the PcstA promoter's characterisation.

Image:II09_M0_C2.jpg


  • Describe briefly purpose behind each test construct:

The purpose of this assay is to characterise the effect of changing between primary and secondary carbon source on the PcstA promoter output.


  • Current status in terms of cloning for each assembly:

Currently attempting ligations.

  • Number of cloning steps remaining before testing construct:

Two ligations.

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

N/A

  • How likely are we to get the cloning finished before [Jamboree - 7 weeks] (deadline to send parts) ? [unlikely, likely, certain]

Very likely.


Data we hope to Get from this

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


Image:Transition.JPG


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

6


Ready for testing construct

  • Protocols written and reviewed?

YES


  • Any expertise needed? Access to specific equipment needed?

Plate reader


  • Modelling done? Ready to analyse experimental data?

No


Colanic Acid & Trehalose Testing

  • Input(s)/Output(s):

In response to a rise in cAMP, the encapsulation phase is initiated. This results in an output of:

1) Trehalose

2) Colanic Acid

3) Membrane-stress proteins

Testing Construct Design

  • How many of them?:

There are four testing constructs.

Colanic acid = 3

Trehalose = 1


  • Describe briefly purpose behind each test construct:

The colanic acid testing constructs are designed to assess the coat's protective potential against acid stress.


The trehalose testing construct is designed to qualitativly show that trehalose is produced.

Progress

Colanic acid constructs:

Image:II09_M2_C12.jpg

The above step is currently being attempted.

Image:II09_M2_C13.jpg
Image:II09_M2_C14.jpg
Image:II09_M2_C15.jpg

  • Number of cloning steps remaining before testing construct:

Once the first ligation has been done, the remaining parts are assembled using SLIC. Therefore, it is not essential to have biobricks for construct assembly, however they are preferable.


Trehalose constructs:

This construct relies on standard biobrick ligations so we are waiting on OtsA and OtsB biobricks before we can start.

Image:II09_M2_C16.jpg
Image:II09_M2_C17.jpg
Image:II09_M2_C18.jpg
Image:II09_M2_C19.jpg
Image:II09_M2_C20.jpg


  • Number of cloning steps remaining before testing construct:

This construct requires 5 ligation steps and so it is estimated that its assembly may not be finished in time.


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

N/A


  • How likely are we to get the cloning finished before [Jamboree - 7 weeks] (deadline to send parts) ? [unlikely, likely, certain]

Colanic Acid

Provided the SLIC goes to plan, I think that we will have the colanic acid testing constructs finished in time.

Trehalose

Of the five ligations, two can be done in parrallel (RBS+OtsA & RBS+OtsB). These parts would be useful to have in the registry. I believe that it is inlikely that the whole construct will be assembled in time for the Jamboree.

Data we hope to Get from this

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


Quantitative analysis of colanic acid coat:

1) Packed Cell Volume = PCV

2) Optical Density = OD

3) Colanic acid produced per cell = PCV/OD of transformed cells divided by PCV/OD of control cells.

4) Percentage of intact cells = PIC

5) Efficiency of colanic acid coat = 4) / 3) (Higher the value the better)


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

10


Qualitative analysis of colanic acid coat:

Electron micrographs:

1) Untransformed Top10 chassis.

2) Transformed with each of the three colanic acid constructs.

3) Chemically induced colanic acid production.


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

8


Trehalose quantification:

This assay will indicate how much trehalose is produced.


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

6

Ready for testing construct

  • Protocols written and reviewed?

Trehalose assay = YES

Colanic acid assay = YES


  • Any expertise needed? Access to specific equipment needed?

Order testing kits for:

a) Trehalose assay kit.

b) Killing stain.


  • Modelling done? Ready to analyse experimental data?

N/A

Comments

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