Exeter/16 July 2013

So what went wrong with our digestion/ligation/tranformation?

So our first try at attaching two BioBricks together didn't work as well as we had hoped! But how do we know if something went wrong with our digestion, ligation or transformation?

We can assume that, as the RFP transformations worked, the problem is probably unrelated to the transformation stage. The digestion and/or ligation are more likely to be the culprits. Most protocols we have received from iGEM headquarters have been looked at by our supervisors, various academics and some of the PhD students we share the labs with, who "optimise" the protocols for the chemicals, enzymes and equipment we use in the lab. This can include changing the chemicals used in each stage, the timings of certain steps, or the equipment we use. However, we did not do this with the digestion and ligation protocols, we simply followed the ones from iGEM HQ.

To double check that it wasn't our transformations that failed, we re-transformed New Parts 1-4 and the Ligation Control. Looking at the plates on 17/7/13, not a single transformation had yielded a colony.

Obviously we didn't have the products from our digestion to hand; they had been used in the ligation steps. But we did have some of the products from our ligation stage, so we decided to run these on a gel to see where we went wrong.

The gel used is a standard TBE gel, made using the instructions found on 8/7/13. The master mix for the gel contained...

• 120 µl distilled water
• 20 µl buffer (includes a dye)
• 5 µl XbaI
• 5 µl SpeI

The XbaI and SpeI should cut our New Parts from their plasmids, so when we run the gel, we should see two bands per lane; one for the plasmid and one for the New Part.

Ligation Gel

It's not normal practice to run a gel of your ligations, but we were bored and upset that our digestion/ligations didn't work...

Considering we cut our plasmids with XbaI and SpeI, which flank our New Part, we would have hoped to have seen two bands on each lane; one for the plasmid and one for the New Part. Looking at the gel, this hasn't happened. We can clearly see where the plasmids are; the line that runs across the gel at ~2.1kb.

For New Part 1 (promoter and RBS added to YF1, our blue light sensor), we would see a band at ~1.2kb to match the New Part, but no such band is visible.

For New Part 2 (promoter and RBS added to FixJ, the protein which communicates between the blue light sensor and the yellow pigment gene), we would see a band at ~680bp. In fact, we see three bands, none of which are ~680bp. We're not quite sure what they are!

New Part 3 (our magenta pigment attached to a terminator) should have a band at ~750bp. This band is not visible on the gel, and we have a strong band at ~2.75kb which is pretty unexpected.

New Part 4 (our yellow pigment attached to a terminator) would have a band at ~730bp, which is not visible on the gel.

The ligation control also appears to have not worked.

Preparing to reattempt the ligations and digestions

We made liquid colonies of...

• 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])

Take me back to the notebook.