Exeter/17 July 2013

MiniPreps of yesterday's liquid cultures

The plasmids were extracted from our liquid cultures, giving us DNA from...

• CcaS, our green light sensor ([http://parts.igem.org/Part:BBa_K592001 BBa_K592001])
• CcaR, the intermediate protein which communicates between the green light sensor and the cI repressor system ([http://parts.igem.org/Part:BBa_K592002 BBa_K592002])
• New Part 2, a promoter and RBS added to FixJ, the intermediate protein which communicates between the blue light sensor and the yellow pigment ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002] + [http://parts.igem.org/Part:BBa_K592005 BBa_K592005])
• Terminator, seven replicates ([http://parts.igem.org/Part:BBa_B0015 BBa_B0015])
• FixJ promoter, where FixJ binds to allow synthesis of yellow pigment ([http://parts.igem.org/Part:BBa_K592006 BBa_K592006])
• Magenta pigment ([http://parts.igem.org/Part:BBa_K592012 BBa_K592012])
• Yellow pigment ([http://parts.igem.org/Part:BBa_K592010 BBa_K592010])
• Ribosome binding site ([http://parts.igem.org/Part:BBa_B0034 BBa_B0034])
• YF1, our blue light sensor ([http://parts.igem.org/Part:BBa_K592004 BBa_K592004])
• FixJ,the intermediate protein which communicates between the blue light sensor and the yellow pigment ([http://parts.igem.org/Part:BBa_K592005 BBa_K592005])

NanoDrop data

We found the concentration of DNA in each sample using a NanoDrop machine. Remember, we had seven replicates of our terminator ([http://parts.igem.org/Part:BBa_B0015 BBa_B0015])

- Terminator #1 - 3.5ng/µl

- Terminator #2 - 1.9ng/µl

- Terminator #3 - 30.5ng/µl

- Terminator #4 - 25.4ng/µl

- Terminator #5 - 2.4ng/µl

- Terminator #6 - 2.1ng/µl

- Terminator #7 - 15.0ng/µl

- CcaS - 94.0ng/µl

- RBS - 40.1ng/µl

- Magenta pigment - 45.5ng/µl

- Yellow pigment - 34.8ng/µl

- New Part 2 (promoter, RBS and FixJ) - 59.5ng/µl

- YF1 (blue light sensor) - 27.6ng/µl

- FixJ - 30.7ng/µl

- FixJ promoter - 20.1ng/µl

- CcaR - 4.9ng/µl

The data from the NanoDrop shows that most of our "important" parts have sufficient concentrations to go on to digestion, but CcaR has too low a concentration to be useful.

To increase the concentration of DNA per µl, the SureClean protocol was utilised. The concentration of CcaR went from 4.9ng/µl to 81.6ng/µl.

SureClean protocol

SureClean is a solution provided by bioline that allows column free nucleic acid purification. After a number of our digests we found the concentrations of resulting DNA to be unacceptably low when measured using the NanoDrop, fortunately SureClean provided us with a method of concentrating our samples.

Protocol:

1 - Add 6 µl of pink co-precipitate to your nucleic acid sample and mix well for 30s. For samples that are greater than 200 µl the pink co-precipitate can be increased accordingly but it isn't ever necessary to use over 20 µl.

2 - Add an equal volume of SureClean to your nucleic acid solution and mix well.

3 - Centrifuge on max speed (14,000 x g) for 10 mins.

4 - Carefully remove supernatant by pipette.

5 - Add a volume of 70% ethanol that is equak to 2x the original sample volume and vortex for 10s.

6 - Repeat step 3.

7 - Remove supernatant and air dry to ensure all ethanol is removed.

8 - Resuspend pellet in the desired volume of TE, water or buffer.

9 - Use NanoDrop to measure concentration of resulting purified sample - hopefully it will have improved!

Second attempt at digestions

Our "Part A" BioBricks will be:

• CcaS
• Magenta pigment
• Yellow pigment
• New Part 2 (promoter, RBS and FixJ)
• YFI (blue light sensor)
• FixJ

Our "Part B" BioBricks will be:

• Terminator #3
• Terminator #4
• Terminator #5

Basically, we're adding terminators to our "important" BioBricks. We have also decided to digest the FixJ promoter and RBS to confirm that the digest protocol is working.

The digestion protocol from 11/7/13 was used, although a few changes were made. We are using FastDigest enzymes, which require less time in the thermocycler at 37^oC (generally 15 minutes maximum). Also, we will run the results of our digest on a gel to show that the enzymes (EcoRI, XbaI, etc) have cut in the right places. This denatures the enzymes, so we don't need the 80^oC stage in the thermocycler.

Running the results of our digestion on a gel

We also tried a different way of running the DNA on the gels. The actual gel itself is the same, and we are still running 10 µl of ladder (HyperLadder 1), but for the actual DNA, we have 18 µl of each Part A/B (listed above) with 3ul loading dye (6X DNA Loading Dye).

We expected to see a line across the gel of the various plasmids the Parts come in, and then a second line for each Part. The terminators, FixJ promoter and RBS

Purifying the DNA from our gel

We expected to see a line of bands running across the gel showing the various plasmids our Parts come in. Each lane would also have a second band representing the Part which had been cut out of the plasmid. The terminators, FixJ promoter and RBS are all very small lengths, so could potentially run off the gel (which appears to have happened in the gel image above).

You can see from the image of the gel that our digestion appears to have worked. We have two bands per lane for CcaS, "New Part 2", FixJ, YF1, magenta, yellow and our RFP control.

We cut the bands which don't represent a plasmid out of the gel using a scalpel, then extracted the DNA from the gel slices using a gel purification kit.

Gel Purification Protocol

• Cut the band of interest from the gel using a razor/scapel. Weigh the slice in a zero-ed Eppendorf; for every 100mg of gel, add 100 µl of binding buffer.
• Incubate the mixture at 50-60^oC for 10 minutes, inverting every few minutes to encourage the gel to dissolve.
• Vortex the tubes. If the solution is yellow, you are free to continue to the next step. If the solution is orange/violet, add 10 µl 3M sodium acetate and mix.
• (The following stage only applies if your DNA of interest is <500bp or >10kb. If the fragment is <500bp, add isopropanol (100mg of gel dissolved in 100 µl of binding buffer requires 100 µl isopropanol). If the fragment is >10kb, add distilled water (100mg of gel dissolved in 100 µl of binding buffer requires 100 µl distilled water).)
• Transfer 800 µl of solution to the column and centrifuge at 13,000 rpm for 60 seconds. Discard the supernatant.
• (If the DNA from this process is going to be sequenced, at this point add 100 µl binding buffer and centrifuge at 13,000 rpm for 60 seconds. Discard the supernatant.)
• Add 700 µl wash buffer, centrifuge at 13,000 rpm for 60 seconds, and discard the supernatant.
• Centrifuge the empty column for 60 seconds on a high speed.
• Transfer the column to an Eppendorf and add 50 µl elution buffer. We only added 35 µl elution buffer, as we are extracting very small amounts of DNA.
• Keep the supernatant. It can be stored at -20^oC.

Take me back to the notebook.