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From 2013.igem.org

Morning

Today we will be trying to digest and ligate some of our BioBricks for the first time!

MiniPrep

We extracted the plasmids for...

- BBa_K608002, a promoter and RBS - BBa_K592005, our FixJ protein intermediate - BBa_K592004, our blue light sensor - BBa_B0015, a terminator

The normal MiniPrep protocol was followed, however the BBa_K608002 and BBa_B0015 Bricks only had 2/3rds as much genetic material collected as the other Bricks. (Plasmids collected from 2 Eppendorfs, as opposed to the usual 3)

Digestions

We are combining...

- a promoter and RBS to the blue light sensor (BBa_K608002 + BBa_K592004)

- a promoter and RBS to the FixJ protein coding region (BBa_K608002 + BBa_K592004)

- the magenta pigment to a terminator (BBa_K592012 + BBa_B0015)

- the yellow pigment to a terminator (BBa_K592010 + BBa_B0015)

First we have to work out the concentrations of each plasmid using a NanoDrop machine.

NanoDrop results

For our promoter and RBS Brick (BBa_K608002) and terminator Brick (BBa_B0015) we have two replicates.

- Promoter and RBS #1 - 29.1ng/ul

- Promoter and RBS #2 - 25.2ng/ul

- Terminator #1 - 27.6ng/ul

- Terminator #2 - 19.2ng/ul

- Blue light sensor - 38.3ng/ul

- FixJ intermediate - 58.1ng/ul

- Yellow pigment - 45.7ng/ul

- Magenta pigment - 72.0ng/ul

- RFP control - 43.4ng/ul

To get a concentration of 25ng/ul for the restriction digest, we will use...

- Promoter and RBS #1 - 17.18ul DNA and 25.82ul distilled water

- Promoter and RBS #2 - 19.84ul DNA and 23.16ul distilled water

- Terminator #1 - 18.12ul DNA and 24.88ul distilled water

- Terminator #2 - 26.04ul DNA and 16.96ul distilled water

- Blue light sensor - 13.05ul DNA and 24.95ul distilled water

- FixJ intermediate - 8.61ul DNA and 34.39ul distilled water

- Yellow pigment - 10.94ul DNA and 32.06ul distilled water

- Magenta pigment - 6.94ul DNA and 36.06ul distilled water

- RFP control - 11.52ul DNA and 31.48ul distilled water

The buffer we are using contains BSA already, so the change from the iGEM Digestion protocol is that 0.5ul BSA has been replaced with 0.5ul distilled water, which is included in the values above.

Restriction digest

Our "Part A" Bricks will be BBa_K608002 (promoter and RBS), BBa_K592010 (yellow pigment) and BBa_K592012 (magenta pigment). Our "Part B" Bricks will be BBa_K592004 (blue light sensor), BBa_K592005 (FixJ coding region) and BBa_B0015 (a terminator).

We will also be using pSB1K3 as our plasmid backbone, so our final ligation products should be kanamycin resistant.

Ligate

• K592010 and B0015
• K592012 and B0015
• K592003 and Q04510
• K592005 and B0015
• K592002 and B0015
• K592001 and B0015
• K592004 and B0015