Team:Cambridge/Diary/Week 10

Monday

A few more people went on holiday and a few came back. Tom ran the gel for the lux-mOrange halves and nothing came out- he discovered that a few fragments we got out during the hectic Thursday and Friday are not of the right size, which might explain why our things are not working. That includes one part of the Ratiometrica vector backbone (vecB), and one part of the lux-mOrange backbone (vecA1). He re-PCRed these using touch-down PCR, and some of it seems to work now so we should start our Gibson again soon...

Tuesday

More PCR, more gels for Tom as he tries to get the correct fragments... He also grew up some overnight cultures with sfGFP-ampR plasmids and will miniprep so we now have more positive control templates.

Wednesday

Emmy did some reorganising and relabelling of all the tubes we've got as there are now too many tubes of DNA fragments for Ratiometrica and lux-mOrage! Tom did some more gel extractions and miniprepped the sfGFP-ampR out of the O/N cultures so now we have positive control again.

LUX-MORANGE PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join the quarters of vector backbone together, and the mOrange and the other half of the vector (re-attempted with correct size fragment)

2a) PCR out each half (Touch-down PCR done... there is severe mispriming and the bands that we want is very very faint, not visible even on some lanes of the triplicates...)

2b) At the same time, attempt a 4-piece Gibson (done, will know tomorrow)

3) Two-piece Gibson the two halves

RATIOMETRICA PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join each of the smaller fragments to the adjacent half of the vector backbone. (re-attempted one of the reactions with correct size fragment)

2a) PCR out each half (only one half came out last Friday, the other half is still not coming out even with the size correction and touch-down)

2b) At the same time, attempt a three-piece Gibson with the half from last Friday, and the two other fragments that are refusing to come out from Gibson (done, will know tomorrow)

3) Two-piece Gibson the two halves

Thursday

Tom re-PCRed a quarter of the vector backbone for Ratiometrica, because we suspect that the size of it from previous PCR was a bit dodgy. Unfortunately there seems to be something wrong with the mastermix- no bands and positive control didn't work. Not to worry- we will try again tomorrow as usual.

Nothing's grown on the plates from last night- not even if the positive control. It might've been a problem with the Gibson protocol. We also tried to carry out gel electrophoresis directly with the Gibson products from yesterday night, unfortunately there are no bands. It might be because there were too little DNA, or it might just be the Gibson not working. We tried the 2 two-piece Gibsons again from yesterday for the luciferase construct, one half has came out very nicely but not the other- probably wrong primers. Will have to do it again tomorrow, as always.

We have devised a new strategy to tackle the problem with Gibsoning Ratiometrica. We planned to together the smaller fragments later tonight, and then attempting a three-piece Gibson with the new fragment and also the 2 backbone fragments. Unfortunately no band of the right size came out from PCR of the Gibson product of the two smaller fragments so we can't do the three-piece Gibson yet afterall. It is not much of a surprise: the fluorescent proteins primers are very similar to each other, and since our desired product has the 2 fluorescent proteins at the beginning and end, it is very likely to have misprimed. We will need to figure out some way around this problem. We also PCR-ed up some more of the smaller fragments as we are running out of DNA.

Also, we are ordering primers to ligate the vector fragments into a closed circle, to be used as a better positive control compared to the sfGFP-pSB4K5 that we have been using. Furthermore we will be looking into standard assembly as a plan B to get Ratiometrica out.

Lastly, Oli began PCRing up the fragments for the construction of the Mg2+ riboswitch construct again, with the new master mix. Unfortunately, the PCR of the vector failed, so he will try that again tomorrow.

And we booked hostel for Amsterdam! (Thanks Charlie!)

Friday

Well, we now have the construct development cycle down to a single day, so long as we have the primers. Oli managed to PCR out the vector DNA, extract it, Gibson it with the fragments from yesterday and transform it into some e.coli.

Tom also began running some verification gels to check that we have the fragments that we thought we had. Turns out, we do. He'll check with PJ tomorrow what he thinks the problem with our Gibson reactions is.

It was Kat's second to last day, so she introduced us to Silvano's (other coffee shops are available). Bizarrely (but kindly), she bought the coffees.

Saturday

One precious colony from the entire Gibson reaction! We're growing it up to test it tomorrow with IPTG and the MG2+ so we can see if it is what we thought it was.

Because of the ludicrously low efficiency of the Gibson demonstrated by weeks of effort, PJ has offered to run a Gibson and transformation in parallel with Tom to see if our master mix or transformations are at fault. We discovered that, for a two fragment reaction such as we have been doing, he usually gets thousands of colonies with only 3 μl of the final transformed cell suspension. We, on the other hand, got about one hundred with 200 μl. Obviously something is going rather wrong.

It could also be that our gel extractions are not very effective, given that the verification gels we've been running show that our DNA solutions are about 2 - 6 ng/μl (as opposed to 10 - 30 that we should be working with). If PJ and Tom get the same results as each other, we'll know that's the problem.

Some of us are beginning to get a bit panicky that it's already September...

Sunday

Well, the good news is that the colonies picked from yesterday seem to be fluorescing, indicating that they have indeed been transformed with the plasmid we had hoped. Unfortunately, they are fluorescing, indicating that the inhibition that should be caused by the lac I repressor is not working. Perhaps there are upstream regions that are vital to the successful function of the repressor, perhaps the riboswitch is messing up its function.

[http://jb.asm.org/content/170/7/3283.abstract Initial search results] indicate that magnesium is a considerable component of normal e.coli growth mediums such as LB broth. It tends to be in the area of 200 μM, well within the theoretical sensitivity range of our riboswitch, possibly explaining the fluorescence. Unfortunately, magnesium is also essential for growth, so we'll have to transform this construct into bacillus before we can properly determine its functionality.

Debugging of the Gibson protocol is also continuing, with Tom trying several different techniques to determine the best way to extract DNA from our gels. Sodium acetate shows initial promise, though more tests will have to be done to determine its efficacy.