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We begin settling in and setting ourselves up in the lab. The adventure starts now! Plenty of paperwork to do and people to check in with before beginning. Safety comes first, and with it safety courses to teach us what to watch out for when working in the synthetic biology lab.
We spend some time discovering the equipment at our disposal, how it works and what it can and can't do. We have access to a microplate reader (absorbance and fluorescence), a spectrophotometer (absorbance), an inverted fluorescence microscope, a PCR machine an, multiple centrifuges and electrophoresis machines, and plenty of pipetmans, plastic disposables and gloves. I'm sure there's a couple of things I've forgotten on the list, but at least we know we have what's necessary to start on great footing.
We have transformed fresh cells with a GFP gene controlled by a PBad promoter using INSERT PROTOCOL NAME HERE
Experimental work begins in earnest!
We have a GFP gene controlled by a PBad promoter, and we want to test this promoter to see if it will be useful in our later experiments with KillerRed, one of the key proteins in our project. We can use the GFP Tristar microplate reader. Production is induced with arabinose, a kind of sugar.
Qualitatively, fluorescence starts appearing three hours after induction with arabinose. Originally we didn't think it would take this long. A microscope picture was taken, where we can clearly see GFP fluorescence. Our advisors say that we didn't have enough controls to make sure this was actually GFP expression, though. THis is true as we didn't compare our results to wild-type cells, and it means we will have to be more rigorous from now on. Examples would be wild-type cell autofluorescence, LB autofluorescence, and an estimation of the measurement errors incurred by our use of devices such as the microplate reader.
As we know the state of the cells is often determined by the amount of cells in a culture when a fresh culture is started, we want to obtain OD600 measurements at the same time as fluorescence measurements on the microplate reader. Unfortunately we can't do this with our microplate reader, since the photomultiplier has a certain position for fluorescence readings and another for absorbance readings. (the user has to manually change the position of the detector every time one wants to change the mode). We could also have problems if we used clear plastic microplates to do fluorescence measurements as well as absorbance measurements since there can be fluorescence crosstalk between adjacent wells.
We thus have to use fluorescence only. A characterization curve has to be made in order to link actual OD600 measurements to their equivalents in fluorescence. The instrument would measure the amount of light scattered by the samples, which is a function of OD, by using the same emission and excitation filters.
The trend seems to be decreasing fluorescence with increasing OD600, contrary to our expectations, as intuition would have it that scattered light increases with the amount of cells. The trend seems linear but this isn't sure because of measurement noise: we would need additional points to make sure. Another experiment has to be done.
Meeting with Yohan Roulland at the Institut Albert Bonniot (Grenoble medical campus), regarding the use of KillerRed. Information obtained: KillerRed is more effective as a dimer, whereas the gene itself only codes for a monomer. The dimer form can be obtained by fusing one KillerRed protein to another directly after it on the gene, or by fusing it with two different proteins which operate close to each other in the cell, thereby bringing their respective KillerRed proteins close enough for dimerization to occur. Expression of KillerRed in the cytoplasm of different organelles in eukaryotic cells hasn’t had any effect: expression in the cytoplasm of the nucleus didn’t successfully kill the cells. Since it is most probable that bacteria are more resistant to oxidative damage than eukaryotes, this would mean that a specific target protein needs to be chosen in E. coli so as to significantly damage the cell and kill it when KillerRed produced ROS. Experiments As the first experiment didn’t yield any quantitative information, we need to develop a rigorous protocol in order to substract background noise from fluorescence measurements more precisely, as well as prove that only the bacteria that have been transformed with the correct plasmid, and induced with arabinose, are actually fluorescing. We have a microplate reader at our disposal that can perform repeated measures at regular intervals as well as injections of liquid at designated times. We can use it to keep the culture warm, shaked and to induce production of GFP with arabinose at a certain time. The experiment consisted in preparing a microplate with different solutions containing a combination of the following: Pbad-GFP transfected cells or Wild-type cells in Luria-Bertani Miller growth medium Arabinose at different concentrations Kanamycin as an antibiotic to select for Pbad-GFP transfected cells
PBAD promoter characterization: we decided to try and characterize the behaviour of PBAD according to the concentration of arabinose in the media. It is more rigorous than the first PBAD experiment we did.
We used E. coli BW25113 transformed with PBAD-GFP (Kanamycin resistance in the plasmid), and untransformed BW25113 as control. Cultures were saturated over the weekedn before the experiment.
We used decreasing concentrations of arabinose ranging from 0.1% to 0.00016% with 5X dilutions.
Results show fluorescence starts rising after 3 hours of incubation on microplates.
In order to obtain better results in terms of fluorescence and in terms of bacterial growth control, we choose to use M9 for a second experiment to characterize PBAD.
M9 is supplemented in glucose. We use E. coli BW25113 which is a prototroph.
The protocol used is available on the internet. The experiment failed for multiple reasons including lack of controls, lack of time to do every step every 30 minutes.
