Exeter/15 July 2013

From 2013.igem.org

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=Cyan pigment and new growth media=
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We've been having some issues getting our cyan pigment (BBa_K322122) functioning. BBa_K322122 is comprised of two sections; HO1 which codes for the enzyme heme oxygenase, and pcyA which codes for the enzyme ferredoxin oxidoreductase. Together these synthesise phycocyanobillin, which is acting as our cyan pigment, but is also required as a part of our red light sensor, cph8. This means we need lots of phycocyanobillin to be synthesised; enough to make our red light sensor functional, and then excess phycocyanobillin to make our cyan pigment.
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 +
So far, we can't seem to get enough phycocyanobillin being made to make the cyan pigment, but we are also concerned that, as heme oxygenase and ferredoxin oxidoreductase both contain Fe ions in their active sites, maybe there is insufficient Fe in our LB agar to allow the enzymes to function well.
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 +
We've decided to make some hemin growth media, which should have more Fe and encourage the cyan pigment to develop.
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'''To make 1mmol hemin solution'''
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 +
- Add 3.26mg hemin powder to 5ml 20mM NaOH
 +
 +
- Dilute to 10uM by adding 100ul 1mM hemin solution to 10ml LB broth
 +
 +
- This can be then made into agar plates.
 +
 +
=Transformations=
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We are transforming the Parts listed below, including our four "New Parts":
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 +
* New Part 1 - BBa_K608002 + BBa_K592004, a promoter and RBS added to YFI, our blue light sensor
 +
* New Part 2 - BBa_K608002 + BBa_K592005, a promoter and RBS added to FixJ, which codes for the protein (FixJ) which communicates between our blue light sensor and the yellow pigment gene
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* New Part 3 - BBa_K592012 + BBa_B0015, our magenta pigment added to a terminator
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* New Part 4 - BBa_K592010 + BBa_B0015, our yellow pigment added to a terminator
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* Ligation Control, contains RFP
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* BBa_K098011, which codes for the protein (OmpR) which communicates between our red light sensor and the cyan pigment gene
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* BBa_R0082, the promoter to which OmpR binds
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* BBa_K592002,which codes for the protein (CcaR) which communicates between our green light sensor and inverter gene which eventually regulates transcription of the magenta pigment gene
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* BBa_Q04510, the cI repressor system, which will switch off the synthesis of our magenta pigment
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We will be using ''E. coli'' DH5α competent cells, and including two RFP controls from the Kit Plates. We are working in two teams to do this transformation, so each team will use an RFP control to check their transformations.
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The standard transformation protocol from 4/7/13 was used, but with New Parts 1-4 and the Ligation Control, 2ul of DNA was used instead of re-suspended DNA from Kit Plates.
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=(Next day, results from transformation)=
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- New Part 1, 0 colonies
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 +
- New Part 2, 1 colony
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- New Part 3, 0 colonies
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- New Part 4, 0 colonies
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- Ligation Control, 0 colonies
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 +
- OmpR (BBa_K098011), 0 colonies
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 +
- OmpR promoter (BBa_R0082), 31 colonies
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- CcaR (BBa_K592002), 30 colonies
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 +
- cI repressor system (BBa_Q04510), 0 colonies
 +
 +
- RFP control #1, 38 colonies
 +
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- RFP control #2, 45 colonies

Revision as of 11:54, 18 July 2013

Cyan pigment and new growth media

We've been having some issues getting our cyan pigment (BBa_K322122) functioning. BBa_K322122 is comprised of two sections; HO1 which codes for the enzyme heme oxygenase, and pcyA which codes for the enzyme ferredoxin oxidoreductase. Together these synthesise phycocyanobillin, which is acting as our cyan pigment, but is also required as a part of our red light sensor, cph8. This means we need lots of phycocyanobillin to be synthesised; enough to make our red light sensor functional, and then excess phycocyanobillin to make our cyan pigment.

So far, we can't seem to get enough phycocyanobillin being made to make the cyan pigment, but we are also concerned that, as heme oxygenase and ferredoxin oxidoreductase both contain Fe ions in their active sites, maybe there is insufficient Fe in our LB agar to allow the enzymes to function well.

We've decided to make some hemin growth media, which should have more Fe and encourage the cyan pigment to develop.

To make 1mmol hemin solution

- Add 3.26mg hemin powder to 5ml 20mM NaOH

- Dilute to 10uM by adding 100ul 1mM hemin solution to 10ml LB broth

- This can be then made into agar plates.

Transformations

We are transforming the Parts listed below, including our four "New Parts":

  • New Part 1 - BBa_K608002 + BBa_K592004, a promoter and RBS added to YFI, our blue light sensor
  • New Part 2 - BBa_K608002 + BBa_K592005, a promoter and RBS added to FixJ, which codes for the protein (FixJ) which communicates between our blue light sensor and the yellow pigment gene
  • New Part 3 - BBa_K592012 + BBa_B0015, our magenta pigment added to a terminator
  • New Part 4 - BBa_K592010 + BBa_B0015, our yellow pigment added to a terminator
  • Ligation Control, contains RFP
  • BBa_K098011, which codes for the protein (OmpR) which communicates between our red light sensor and the cyan pigment gene
  • BBa_R0082, the promoter to which OmpR binds
  • BBa_K592002,which codes for the protein (CcaR) which communicates between our green light sensor and inverter gene which eventually regulates transcription of the magenta pigment gene
  • BBa_Q04510, the cI repressor system, which will switch off the synthesis of our magenta pigment

We will be using E. coli DH5α competent cells, and including two RFP controls from the Kit Plates. We are working in two teams to do this transformation, so each team will use an RFP control to check their transformations.

The standard transformation protocol from 4/7/13 was used, but with New Parts 1-4 and the Ligation Control, 2ul of DNA was used instead of re-suspended DNA from Kit Plates.

(Next day, results from transformation)

- New Part 1, 0 colonies

- New Part 2, 1 colony

- New Part 3, 0 colonies

- New Part 4, 0 colonies

- Ligation Control, 0 colonies

- OmpR (BBa_K098011), 0 colonies

- OmpR promoter (BBa_R0082), 31 colonies

- CcaR (BBa_K592002), 30 colonies

- cI repressor system (BBa_Q04510), 0 colonies

- RFP control #1, 38 colonies

- RFP control #2, 45 colonies

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