July

Week 1

Monday

- Present our project to the American Summer School (poster) at Maison Minatec in Grenoble.
- Find a way to draw the spectrum of the wratten filter #55 for Killer Red and green sensor - measure the absorbance of the filter with all the filters available of the luminometer - by tapping the filter to a 96 well plate - We need to find a better and more efficient solution to draw these spectrum.
- Try to preculture cells with pJT122 (Voigt 1) and pPLPCB(S)(Voigt 3) but didn’t grow, so re-transform BW with pJT122, pJT106B(Voigt 2) and pPLPCB(S).

Tuesday

- Use luster terminals to avoid the use of crocodile clip and have longer cable for the highpower LED.
- Test the system where the LED is put in the incubateur and measure the temperature of the surface of a Petri dish exposed to the light of the LED that is 7cm above the dish - conclusion: no overwarming - this is a very good point! If the cells expressing KillerRed died, it would not be because of the overwarming of illumination but because of the ROS emission!
- Put 3 insulated clones of each Petri dishes that contain the transformed cells in order to get a glycerol stock and to do a mini prep in 1mL.

Wednesday

- Do a glycerol stock of 1.5mL for each clone that grows overnight. Use 500µL of the 1mL preculture for a bigger culture (10mL) to do a miniprep and the liquid that left is used on a new Petri dish.
- Do a miniprep but since the Voigt’s plasmid are low copy, I use 4 Eppendorf for 1 columns to get more concentrated DNA.
In the end, there are more than 20ng/µL for all the plasmid. For others colonies, there are more than 100ng/µL - it may be contaminated with chromosomal DNA.

Thursday

- Transform all the cells that already contain one of the Voigt’s plasmid with one or two of the others Voigt’s plasmid.

Friday

- The transformations work more or less for all of them but there is only one colony for the transformed cells with 3 plasmids
- Put three clones of each Petri dish in 3 mL of LB medium to make them grow overnight with the appropriate antibiotics

Week 2

Monday

- Draw a first draft of the Printed Circuit Board (PCB) for the photodiode
- Try to draw the absorbance spectrum using the spectrometer of the Wratten filters ( 55 and 65A), manage to do number 55 but the machine freezes when I measure number 65… it is weird!
- Preculture cell containing Voigt 1&3 and Voigt 1&2&3.

Tuesday

- Try one more time to draw the spectrum of the filter but it seems that for Wratten 65A the scattering light is too low and freeze the machine.
- Draw the absorbance spectra for Simon and Adrien's experience of the BW containing KillerRed after different exposure time.
- Put cells containing Voigt 1&3 on Petri dish with 0.5x of Cm and Spec and 1x of Xgal, then put it in aluminium paper and cut out a square and illuminate it with green light overnight (test of photosensitive promoter)

Wednesday

- Take pictures of the Petri dish with the green sensor, the blue color is not equally distributed. It seems that Xgal is not evenly spread and that there are not enough cells.
- Finish the 2 PCB for the project and send it to an electronic teacher.
- Draw the absorbance spectra for Simon and Adrien's experience of the M15 containing KillerRed after different exposure time.

Thursday

- Think of a way to put GFP under the PcpcG2 promotor in pJT122 instead of LacZ- put restriction site (BglI) at the beginning and at the end of KR ? (because BglI cut LacZ twice at the beginning and at the end of the gene) - Think of the design of our device

Friday

Week 3

Monday

- Put Voigt 1&3 in liquid culture with 4x Xgal ON

Tuesday

- Put the liquid culture of Voigt 1&3 under microscope - notice blue pigment in bacteria but don’t know if it is because of LacZ or because of the irisation of the light

Wednesday

- Evolution of the MATLAB model :
integration of the folding time of KillerRed
-todo in the MATLAB model :
integrate the defence of cells against ROS (sub a quantity of ROS)
integrate the recovery time of the cell (% of cells unable to divide ?)
-stuff I could need to know about a cell to go on, write it below folks :
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- Mr. Bucci answers and gives his point of view on the electronic circuit, some problem because of the low intensity delivered by the system and the fact that the transistor BDX33C is a darlington transistor - means that it is composed by two transistor - consequently Vbe(BDX33C) = 1.4V =2*0.7V= 2*Vbe(classical transistor). Need to redo the calculation

Thursday

- Find a solution for the electronic circuit, since the apply voltage is 12V and the nominal power of the lamp is 6W the intensity is 0.5A. Instead of the potentiometer we will use two resistors in parallel to avoid burning them. And to have 0.7V on these resistors we need to add one diode on the other branch
- draw new PCBs according the the chosen solution
- Think of a way to put KR and RFP under red sensitive promoter - restriction/ligation seems to be easier here

Friday

- Print the 2 PCBs and drill holes into them and weld the different component on them.
- Test the PCB that control the intensity of the light with a function generator and the oscilloscope, it works well.
- Notice that the connector on the photodiode PCB is not welded on the right side

Week 4

Monday

- Weld the connector on the photodiode PCB on the right side
- Design the different primers for the new constructions (KR and mRFP in pJT106b under the red sensor)
- Test the photodiode PCB with the oscilloscope and Arduino, it works like on the workbench =)
- Test the PCB that control the intensity of the light with Arduino, something goes wrong after 3 sec, Arduino freezes and seems to receive order from the computer whereas the computer doesn’t send any order... the oscilloscope seems to indicate that the noise could be the problem || The solution : put a condensator between PWM and GND ?

Tuesday

Wednesday

Thursday

Friday