Exeter/15 July 2013

Cyan pigment and new growth media

We've been having some issues getting our cyan pigment ([http://parts.igem.org/Part:BBa_K322122 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 using standard protocol.

Liquid cultures

New liquid cultures of BBa_K322122 were also made to allow further investigation into producing a cyan pigment tomorrow.

Transformations

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

• New Part 1 - [http://parts.igem.org/Part:BBa_K608002 BBa_K608002] + [http://parts.igem.org/Part:BBa_K592004 BBa_K592004], a promoter and RBS added to YFI, our blue light sensor
• New Part 2 - [http://parts.igem.org/Part:BBa_K608002 BBa_K608002] + [http://parts.igem.org/Part:BBa_K592005 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 - [http://parts.igem.org/Part:BBa_K592012 BBa_K592012] + [http://parts.igem.org/Part:BBa_K608002 BBa_K608002], our magenta pigment added to a terminator
• New Part 4 - [http://parts.igem.org/Part:BBa_K592010 BBa_K592010] + [http://parts.igem.org/Part:BBa_B0015 BBa_B0015], our yellow pigment added to a terminator
• Ligation Control, contains RFP
• [http://parts.igem.org/Part:BBa_K098011 BBa_K098011], which codes for the protein (OmpR) which communicates between our red light sensor and the cyan pigment gene
• [http://parts.igem.org/Part:BBa_R0082 BBa_R0082], the promoter to which OmpR binds
• [http://parts.igem.org/Part:BBa_K592002 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
• [http://parts.igem.org/Part:BBa_Q04510 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, 2 µl 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

Take me back to the notebook.