Exeter/11 July 2013

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

MiniPrep

We extracted the plasmids for...

• [http://parts.igem.org/Part:BBa_K608002 BBa_K608002], a promoter and RBS

• [http://parts.igem.org/Part:BBa_K592005 BBa_K592005], our FixJ protein intermediate

• [http://parts.igem.org/Part:BBa_K592012 BBa_K592004], our blue light sensor

• [http://parts.igem.org/Part:BBa_K592012 BBa_B0015], a terminator

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

NanoDrop and 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_K592005)

- 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 - 29.1 ng/µl

- Promoter and RBS #2 - 25.2ng/µl

- Terminator #1 - 27.6ng/µl

- Terminator #2 - 19.2ng/µl

- Blue light sensor - 38.3ng/µl

- FixJ intermediate - 58.1ng/µl

- Yellow pigment - 45.7ng/µl

- Magenta pigment - 72.0ng/µl

- RFP control - 43.4ng/µl

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

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

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

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

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

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

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

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

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

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

The buffer we are using contains BSA already, so the change from the iGEM Digestion protocol is that 0.5 µl BSA has been replaced with 0.5 µl 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.

Method:

-Labelled PCR tubes with Part A, Part B, pSB1K3 and RFP control (Parts A and B described above)

- Add correct amounts of distilled water and DNA (see "To get a concentration of 25 ng/µl for the restriction digest, we will use...")

- Add 5 µl of 10X FastDigest buffer (which contains BSA) to each tube.

- To the Part A tubes, add 1 µl EcoRI and 1 µl SpeI.

- To the Part B tubes, add 1 µl XbaI and 1 µl PstI.

- To the pSB1K3 tube, add 1 µl EcoRI and 1 µl PstI.

- To the RFP control, add 1 µl EcoRI and 1 µl PstI.

- Mix all by pipetting up and down gently. DO NOT VORTEX. Flick the tubes to collect the liquid at the bottom.

- Incubate in a thermocycler - 37 °C for 30 minutes, then 80 °C for 20 minutes. As a precaution, the final temperature was set at a 4 °C infinite hold, incase we didn't get to the thermocycler immediately when the cycle finished.

Ligation

We need...

- PCR tubes

- distilled water

- T4 DNA ligase reaction buffer

- T4 DNA ligase (MUST be kept in the freezer until last minute)

- pSB1K3 from digest

- Part A from digest

- Part B from digest

- RFP control from digest

Method:

- Label a PCR tube as "New Part" (New Part 1 is promoter and RBS + blue light sensor, New Part 2 is promoter and RBS + FixJ intermediate, New Part 3 is magenta pigment + terminator, New Part 4 is yellow pigment + terminator)

- To these tubes, add 2 µl pSB1K3, 3.3 µl Part A, 3.9 µl Part B, 1.0 µl T4 DNA ligase reaction buffer and 0.5 µl T4 DNA ligase.

- Mix gently by pipetting up and down. DO NOT VORTEX.

- Also label a PCR tubes as ligation control. This will contain 2ul from the RFP control digest, 6.5 µl purite water, 1 µl T4 DNA ligase reaction buffer and 0.5 µl T4 DNA ligase. Mixed gently by pipetting up and down.

- Collect liquid at bottom of all PCR tubes by flicking.

- Incubate in the thermocycler for 30 minutes at 16 °C then 20 minutes at 80 °C. As a precaution, the final temperature was set at a 4 °C infinite hold, incase we didn't get to the thermocycler immediately when the cycle finished.

Our products from ligation were stored overnight at -20 °C to be worked with tomorrow.

Take me back to the notebook.