Team:Cambridge/Diary/Week 13

Monday

• Ratiometrica: grow O/N cultures for rough IPTG induction tomorrow, ordered primers to convert into biobrick

Tuesday

• Ran PCR for making SPOBB biobrick, gel yielded insufficient results. Redesigned and reordered primers
• Ratiometrica: rough IPTG induction with 8 different concentrations of IPTG, after 3 hours of incubation there was unfortunately no obvious results (i.e. no visible difference between different concentrations/induced and uninduced smaples), will leave them growing overnight and observe again tomorrow; sequencing primers arrived so the plasmid is sent off to sequencing, will have results tomorrow morning; more O/N cultures grown for miniprep to put into Bacillus tomorrow.

Wednesday

• Ratiometrica: Sequencing results came back- good news: CFP is inserted correctly after pSPAC, bad news: more uncertainties as to whether the K143053 and YFP are inserted correctly. Since the cells shows clear YFP under the fluorescent microscope, we cannot really tell what is wrong at the moment, more sequencing primers are ordered to verify the sequence further along the construct. Tom ran a restriction digest with the new miniprep products and they showed exactly the expected results- so we are now more certain that we have got the right construct! IPTG induction in liquid cultures yesterday really didn't give reliable results, so we have now plated cells on IPTG plates for induction and hopefully we will see results tomorrow. Bacillus has also been transformed- it's been a while since we've done Bacillus transformation, and we will know if it works tomorrow. Primers arrived today morning and PCR has been done to convert ratiometrica into a biobrick! We are having some slight problems with the linearised backbone but are slowly working our way around it... hopefully it will all come into place.

Thursday

• Ratiometrica: Yesterday's IPTG induction on plates seemed to have worked! Cells on IPTG plate showed much more CFP than those on the uninduced plates. We are growing O/N cultures in minimal medium so we can test them on a plate reader to get more accurate results and to characterise the part... We are still having trouble with the linearised backbone, plan A is that this currently running PCR cycle will work out and we will have the suitable backbone, plan B is to wait for the correct primers to hopefully come in asap! Bacillus transformation from yesterday produced 1 bacillus colony- it is hard to tell if it is the thing we are looking for as ECFP and EYFP signals are rather poor in Bacillus without the ComGA codons, but there is definitely a little bit of CFP and YFP. We have grown O/N culture of that in LB and will probably extract the genomic DNA then PCR as a verification test. Time is running out...

Friday

• Ratiometrica: we just found illegal restriction sites in our construct ): It is behind the inserted promoter- that is why we didn't notice it in the first place, anyway, it means that we will have to wait for the correct primers to arrive to convert the construct into a biobrick. Meanwhile, M9 minimal cell cultures seem to take double the time to grow so we have made more so they could grow over the weekend and we will have them ready for a plate reader assay by Monday. M9 plates are made as a plan B, so if the plate reader assay doesn't work out we could still photograph plates under the fluorescent microscope as a proof for a working construct. We pelleted the ratiometrica B. sub and it seems to be fluorescing, though as usual it is hard to tell if it is only internal fluorescence... we will attempt a colony PCR tomorrow to verify.

The biobrick fragment was successfully pcr'd out of a Ps3393 miniprep using half prefix/suffix biobrick primers in order to ligate this with the standardised backbone.

Saturday

• Ratiometrica: Cloudy M9 culture! Which means cells take around 48 hours to grow in M9 minimal media. Some of that culture was plated onto M9 plates and we should hopefully be able to check for fluorescence tomorrow. Colony PCR is performed on the Bacillus culture and guess what- no bands. It is likely to be a colony PCR fail though (as they always do) as positive control is not working either. We might have to use some alternative ways to test it out...

• Sporage and distribution: Succesfully extracted the standardised backbone from a lux-biobrick miniprep. This was done in such that the restriction enzymes had enough base pairs to grab onto to cut at the speI and XbaI sites (rather than using the linearised backbone). This was then used with the spoVAA fragment for a ligation reaction and transformation.

Sunday

• Ratiometrica: No colonies on M9 minimal medium plates after 24 hours- these cells really don't like the lack of nutrients. We will give them another day. Same goes to the cultures which have been in the 37°C incubator, they only reached an OD600 of 0.004 after 48 hours. Luckily we have some cultures from Thursday afternoon which are nice and cloudy, so we will be using that (diluted 5 times) for tomorrow morning's plate reader assay. Checked the suspected B. subtilis under the fluorescent microscope again... they don't look very fluorescent unfortunately. Maybe we haven't got it afterall. We will try transforming some again...

• Sporage and distribution: Realised that the spoVAA fragment had not been restricted before ligation so this experiment was doomed to fail. However plenty of colonies were found that (after checking with a restriction digest) were proven to just be the PSB1C3 backbone. This took place due to the natural elimination when XbaI and speI sites are run through a restriction/ligation protocol.