Week 8

Our transformations yielded colonies, but there were much more than expected on our vector control plates. We decided to PCR screen 4 colonies from each of the the ligations. After running them on a gel, it looks as though some of these colonies were successful ligations! At the end of the day, we cultured E0240+I13453 so that we could proceed with more construction tomorrow.

We have mutants! At least, they appear to be - our mutant promoters are in front of RFP, and our colonies appear varied. Loading them into the Tecan for quantitative fluorescence measurement; perhaps we can use RFP for some preliminary data!

After miniprepping the cultured E0240+I13453, we set up a fast digest such that we could try a triple ligation. Our goal parts were: E0240+I13453+C0040, E0240+I13453+C0012, and E0240+I13453+C0051. We cut both E0240+I13453 and our repressor sequences as inserts and ligated them into a pSB1C3 backbone. We transformed and plated the ligation products.

PCR reaction on GFP didn't work (no bands). Will try again.

Our triple ligation plates don't look very good. Most plates only have one colony while our vector control (pSB1C3) has about three. Today, our plan is to attempt to construct the same parts but by using our usual approach with fast digest enzymes. We digested, ligated, transformed, and plated these parts. Hopefully tomorrow we will see better results.

PCR reaction on GFP worked! We digested the product and set up for ligation tomorrow.

Also, we began to work with the Tecan to perfect our measurement protocol. We seem to be picking up a large amount of our excitation wavelength from the machine when we do an emission scan - is this normal?

Once again, our plated transformation were not ideal. The vector controls revealed that there was quite a bit of background on our plates, so we decided to PCR screen colonies once again. We were able to get a few good colonies from this screen, but overall it was not very successful. This lead us to try digesting, ligating, and transforming once more. We will probably stop attempting to use fast digest enzymes, as over the past few weeks they have given us unsuccessful results. Additionally, while digesting with normal restriction enzymes, we will try to allow them to incubate for much longer than three and a half hours (up to 7).

After thinking about it, picking up some of the excitation light in the emission is fine - we can simply subtract it out using a blank.

Ligated and transformed GFP mutants behind an IPTG-inducible promoter (J04500). This should help us assess the mutation efficiency of our protocol.

In the morning we set up a normal digestion and will purify, ligate, and transform it on Sunday.

Lots of GFP mutants! They look like they vary but it's hard to tell from the colonies. We'll streak them out and see how different they really are.