iGEM 2009 — Team Cambridge

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# Team:Cambridge/Notebook

### From 2009.igem.org

please use the new links on wiki.

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| Contents   * [1 iGEM Thought-shower](#iGEM_Thought-shower)   + [1.1 Monday 12 July](#Monday_12_July)   + [1.2 Tuesday 13 July](#Tuesday_13_July)   + [1.3 Wednesday 14 July](#Wednesday_14_July)     - [1.3.1 Colour Wheels](#Colour_Wheels)     - [1.3.2 Viable Pigments](#Viable_Pigments)     - [1.3.3 Follow ups for tomorrow](#Follow_ups_for_tomorrow)   + [1.4 Thursday 15 July](#Thursday_15_July)     - [1.4.1 Pigments](#Pigments)     - [1.4.2 Inputs](#Inputs)     - [1.4.3 Population Control](#Population_Control)   + [1.5 Friday 16 July](#Friday_16_July) * [2 The Project](#The_Project)   + [2.1 Monday 20 July](#Monday_20_July)   + [2.2 Tuesday 21 July](#Tuesday_21_July)   + [2.3 Wednesday 22 July](#Wednesday_22_July)   + [2.4 Thursday 23 July](#Thursday_23_July) |

## iGEM Thought-shower

### Monday 12 July

Each team member researched their own ideas for our project

### Tuesday 13 July

Afternoon presentations - possible projects included

* Bacto-Stat
* Counterfeit Bill Detector
* Traffic light-obedient bacteria
* Light-induced pigment production
* Predator prey bacteria + a parasite
* Modelling pesticide resistance
* Galvanotaxis
* Wave-pulse bacteria
* Ai2 quorum sensing system

**Conclusions**

Decided to concentrate on a pigment output. Wednesday plans involve discussing ideas for pigments, following the same meeting pattern used on Tuesday. Ideally the pigment output would be a new registry part which could then be connected to any input system

### Wednesday 14 July

Investigated possible outputs, with a kind of bacterial printer in mind.

#### Colour Wheels

* Primary school style: BLUE, YELLOW, RED
* True colour wheel: YELLOW, CYAN, MAGENTA
* Autochrome: ORANGE, GREEN, VIOLET

#### Viable Pigments

* In an ideal system, we would have lots of pathways making different coloured pigments from a common precursor. One can dream...

* Carotenoids - RED, ORANGE, YELLOW
  + Biobricks exist for part of the system
  + apparently you can go from yellow to white (cool)

* *Pseudomons aeruginosa* - RED, GREENISH/BLUE
  + Pyocyanin is greenish/blue, can be synthesized from chorismic acid, or more simply, from phenazine-1-carboxylic acid (PCA)
  + Knocking out one of the genes between PCA and pyocyanin leads to the production of a red pigment

* *Chromobacterium violacein* - VIOLET, CYAN
  + Violacein is a violet pigment
  + A precursor is cyan.

* BROWN
  + melanin - easily attainable
  + anyway we could make black?

#### Follow ups for tomorrow

* Possible inputs
* Population control
* bacterial chlorophyll?

### Thursday 15 July

Hoping to start wet work on Monday!

#### Pigments

* Duncan has orange and brown bacteria we can start to work with. The melanin gene has been sequenced, has a restriction site we would need to remove to submit it to the registry.
* We have the genes for violacein! However there seem to be several internal restriction sites within the plasmid that would need to be removed, with three being within the violacein operon.
* Contacted the authors of a paper to get the genes for pyocyanin biosynthesis. Two of them have forbidden restriciton sites, so we'll need to figure out how to remove them.

#### Inputs

* explored the idea of using common repressor / inducer systems - arabinose, lac repressor, and tet repressor - to control pigment production
* scourged the registry for lots of different inducible promoters

#### Population Control

* Crispian proposed a growth control dependency pathway that would make the growth of each type of bacterium (red colour-producing, blue colour-producing, yellow colour-producing, for example) dependent on one other using HSLs

### Friday 16 July

* found where to get PCA, pyocyanin precursor
* did an inventory of supplies
* familiarized ourselves with protocols and planned for next week's wet work
* Thinking about a team logo

Current plans for the wet work involve starting transformation of the bacteria in order to view the pigment colours and intensities. For this we will need to create competant cells and then use Duncan's plasmids to transform the violet and brown and the biobrick registary parts to transform the red and orange.

## The Project

### Monday 20 July

Finalized our project into a few **objectives**

* Pigment production
  + Transform E. coli to produce pigment
  + Hook up the pigmentation genes to inducible promoters
* Shutter mechanism
  + build, test a shutter mechanism
  + Hook up pigmentation genes under inducible promoters to the shutter mechanism
* Improve on previous iGEM projects
  + arsenic sensor?
  + bacterial photography?
* Modelling
  + shutter mechanism
  + Growth rate regulation

**Goals for Wet Work**

* Inducible pigment production integrated with a shutter mechanism
* Possibly attach the arsenic bio-sensor to our green/red pigment output

### Tuesday 21 July

**Proceedure to make competant cells started**

* One colony of TOP10 *E. coli* added to 50ml of LB broth. Incubated overnight in the shaking incubator

**Planned and prepared for transformation**

* Media made up
  + Two litres of 0.1 mM HEPES
  + One litre of LB broth (for making competant cells)
  + 100ml of 10% glycerol
* Ampicillin made up with water to make 8 x 1ml aliquots of 100mg/ml
* 15 LB agar and 100ug/ml Ampicillin plates made up

**Dry work**

**Plan for tomorrow**

* prepare trimethoprim stock solution
* make LB agar and trimethoprim plates as well as LB agar and copper + tyrosine plates
* Complete proceedure for making competant cells (transfer bacteria into big flask, wash with HEPES, and aliquot into glycerol)
* Attempt transformations by electroporation
  + pPSX plasmid (violet)
  + melA plasmid (brown)
  + biobricks

### Wednesday 22 July

**competent cell procedure**

* completed (see protocol page for details of the procedure)

**planned and prepared for electroporation tomorrow**

* made up 4 LB agar and 100ug/ml Ampicillin, 0.5ug/mL tyrosine, 15 ug/mL CuSO4 plates
* plan to transform TOP10 competent bacteria with
  + melanin plasmid, pTRCmelA plasmid --plate onto tyrosine/copper/ampicillin plate
  + violacein plasmid,Plasmid pPSX-Vio+ -- plate onto trimethoprim plate
  + orange biobrick, BBa\_K152005 -- plate onto ampicillin plate
  + promoter, R0011 -- plate onto ampicillin plate
  + more?
* need to make trimethoprim stock solution 10mg/mL acetone, and plate at 50ug/mL
  + tried to make the stock solution today, couldn't get the solid trimethoprim to dissolve in acetone
* need to work out exact volumes of plasmid to be added to the cell (concentrations written on the side of the bottles)

### Thursday 23 July

**Electroporation**

* transformed Top10 competent cells, incubated the following plates:
  + Top10 + pTRCmelA plasmid on tyrosine/copper/ampicillin plate
  + Top10 + Plasmid pPSX-Vio+ on trimethoprim plate
  + Top10 + BBa\_K152005 on ampicillin plate
  + Top 10 + R0011 on ampicillin plate
  + 1:10 dilutions of each as well

| [Home](/Team%3ACambridge "Team:Cambridge") | [The Team](/Team%3ACambridge/Team "Team:Cambridge/Team") | [The Project](/Team%3ACambridge/Project "Team:Cambridge/Project") | [Parts Submitted to the Registry](/Team%3ACambridge/Parts "Team:Cambridge/Parts") | [Modelling](/Team%3ACambridge/Modelling "Team:Cambridge/Modelling") | **Notebook** |
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