June

Week 1 (3-7)

Monday

We observed that absorbance measurements differed according to the device used to make them. For example, the Tristar microplate reader and the Thermo Electron Corporation Genesys 6 spectrophotometer we were using gave different OD600 readings for the same samples. This can be explained by the fact that OD600 measurement values come from diffusion rather than actual absorbance, and thus the distance from the sample to the detector is a factor in the reading. Since it is likely these distances are different from one machine to the other, it would explain the differences in measurement.

We are then confronted with a dilema: which machine do we trust when referring to literature that mentions OD600 values? We settle that an OD600 reading corresponds to a concentration of bacteria in a culture. We then need to establish the relationship between this concentration and the OD600 reading for both machines. For this we will use cell counting chambers and a microscope to count the cells in a small sample volume. Multiple readings will allow us to eliminate the significant noise that stems from such a measurement. The target is around 100 cells per counting chamber in order to get statistical significance without having to count too many cells.

We know that the OD600 curve is linear up to A=0.5 . With values beyond this threshold, the curve levels off and measured values are lower than actual values.

Tuesday

Preparation of freezable competent cells (M15): 40 alicos of 100µL
Test of the competent cells.

Wednesday

Beginning of ‘intra-cell ROS concentration’ modelisation.

Preparation of competent cells via the TSS method:
This method gives cells that are more competent than with CaCl2, but the reagents are harder to prepare. The cells can be stored at -80°C like CaCl2-competent cells.

2X TSS composition is:

10% in volume DMSO 20% in mass PEG-6000 or 8000 MgSO4 at 100 mM concentration

in Lysogeny Broth (Luria Broth) at final pH=6.5

Transfection of competent cells with the biobricks :
- pLac
- mCherry
- cl Inverter
- RBS (Elowitz 1999)
- GFP

First contact with Arduino : the electronic device that will be used as a controler, just as the Cambridge 2010 iGEM team.
Culture of the BBs k174000 and K592004/5/6

Thursday

Failure with the transfections.
Other problem with the petri dishes : everything grew, even the WT colonies that should have died because of antibiotics. Maybe the antibiotics are too old since they were used by Grenoble iGEM Team of 2012.

Another note: since M15 cells were used last time and contain the pREP4 plasmid, this plasmid may prevent isolation of other plasmids of similar sizes during electrophoresis after minipreps with transformed M15 cells. It is then useful to make a stock of competent wild-type cells without extra plasmids. Extra care must be taken when preparing these types of competent cells since there is no antibiotic in the solution, and so contaminations are more likely to happen.

Used cells were BW25113.

Note: cells were vortexed during the preparation to resuspend them but this is apparently not recommended at all! They may have been damaged by this treatment and thus the preparation will have to be made again.

Friday

Preparation of freezable competent cells (WT)(40 alicos of 100µL)

The protocol is available on the team wiki.

The cells used were M15 cells featuring the pREP4 lacI-producing plasmid (M15 cells are an expression strain used to express recombinant proteins from the lactose promoter. LacI prevents expression until the researcher introduces IPTG into the culture. Since most strains do not produce enough lacI for good repression, this plasmid makes more so that repression is efficient). The pREP4 plasmid confers kanamycin resistance.

Same transfections than wednesday, with fresh antibiotics.

PCRs of pAraBad, mCherry and KillerRed, only pAraBAD worked.

Team meeting
TODO List :
- Find a name
- Begin construction of :
->pLac-RBS-YF1-fixJ
->pFixK2-RBS-GFP
- glycerol-stock BBs
- E-glometer, caracterisation of LED

Week 2 (10-14)

Little Summary with Keywords

Monday

PCRs of mCherry and KR (did not work). Is there a problem with the primers or with the DNA templates? Need to work on that
Put the transformed cells on petri dishes with the right antibiotics
Receive the TSL230RD photodiodes
Begin the test of Arduino (digital output) by turning on and off a LED and using a Graphical User Interface (GUI) to control the intensity.

Construction of the pQE30::KillerRed vector

Incubated a 50mL overnight culture of E. coli M15[pRep4] cells (Qiagen, Venlo, Netherlands), containing the pQE30::αSNAP vector, in preparation for midiprep.

Several of our cultures show signs of contamination with different organisms than E. coli. This is very detrimental to cultures as there can be contamination of DNA in minipreps, the cultures don't grow as well if there is competition and it makes physiological studies of the bacteria impossible.

One of the contaminated cultures shows antibiotic resistance that it shouldn't have. This is perhaps caused by bad antibiotics that have degraded with bad storage conditions. The antibiotic stocks for ampicillin, a beta-lactamase-type antibiotic that is particularly prone to degradation over time, were replaced.

Tuesday

Construction of the pQE30::KillerRed vector
Midi-prepped pQE30::αSNAP. The DNA sample concentration was only of 10 ng/µL. The experiment has to be re performed.

Incubated a 10mL overnight culture of E. coli M15[pRep4-pQE30::αSNAP] cells, in preparation for miniprep.