Part:BBa_K3788026:Experience

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Quantitative measurement of fluorescence

To follow the fluorescence intensity’s variations across time of our bacteria, we induced the protein production in liquid culture with different concentrations of Arabinose.

The first step was to prepare 10mL starters of bacteria containing our plasmid on the day before to allow bacterial growth overnight. In the morning, we measured the 600OD of our starters and then took a volume equal to 600OD=0,05 in a final volume equal to 100mL in 2 different Erlenmeyer. Once the 600OD is higher than 0,2 in one of the two Erlenmeyer, we take 10mL that we put in another Erlenmeyer (7 times) and add a certain quantity of Arabinose and then let it incubate at 37°C.

Once the 600OD is higher than 1 in the other Erlenmeyer, we did the same manipulation as before.

Table : Concentrations of arabinose in a final volume of 10mL, used for induction of protein expression

We measured the fluorescence every 60 minutes during 5 hours with the TECAN machine, a 96-well plate reader.

Figure :Schematic representation of the protocol followed to measure the Arabinose induction

Figure : Fluorescence intensity across time in different conditions of induction with adding of arabinose at ₆₀₀OD=0,2

Figure : Fluorescence intensity across time in different conditions of induction with adding of arabinose at ₆₀₀OD=1

Both figures show that the highest level of fluorescence is obtained with Arabinose induction 1%.

However, the fluorescence intensity levels measured are very low, compared with BBa_K3788013 fluorescence intensity levels, which may be due to a low protein expression that could be a problem for further immunodetection.

Visualization with a confocal microscope

To help us determine if the fluorescence is located in the periplasm or in the cytoplasm, we used a confocal microscope to visualize our cells.

In order to do that, we prepared 3mL 2YT growing medium with ampicillin and then put MG1655 bacteria containing either empty pBAD24 (induced with 1% arabinose) or pBAD24 with BBa_K3788026 plasmids (induced with 1% arabinose/non-induced). After 3 hours of incubation at 37°C, we prepared the plates and visualized our cells.

Figure : Visualization with a confocal microscope of bacteria containing pBAD24 with BBa_K3788026 induced with 1% Arabinose (left); pBAD24 with BBa_K3788026 no induced (middle) or empty pBAD24 (right)

We can see that bacteria containing pBAD24 induced with 1% arabinose, and bacteria-containing pBAD24 with BBa_K3788026 without induction do not emit light, but the bacteria containing pBAD24 with BBa_K3788026 with 1% arabinose induction does emit light under a confocal microscope.

However, we are not able to determine whether the light is located at the surface of the bacteria or if it is located in all the cells. We can’t conclude about the localization of our protein.

immunodetection

To ensure the localization of our protein we made an immunodetection with anti-GFP antibodies. As AidaI has an proteolytic activity, our sfGFP is supposed to be in the extracellular medium.

To detect the GFP with antibodies we also centrifugated our cells and charged the supernatant in the SDS-PAGE.

Figure : Nitrocellulose membrane showing the results of the immunodetection led on cells and supernatant of bacteria containing pBAD24 and BBa_K3788026 induced or not.

Controls: bacteria containing empty pBAD24, induced with 1% arabinose for 5 hours; charge control for anti-GFP antibodies.

Rule page Ladder molecular weights (kDa) (lowest to highest band) : 10 ; 15 ; 25 ; 35 ; 55 ; 70 ; 100 ; 130 ; 250

We can observe a band at a molecular weight between 25 and 35 kDa; according to ApE, sfGFP protein has a molecular weight equal to 26,8 kDa, so we could think that this band corresponds to sfGFP that has been cleaved from Aida-I autotransporter, but not secreted.

However, we know that the protein transported to the periplasm with the Sec pathway doesn’t take their final conformation before being in the periplasm, so we can make a different hypothesis :

-Our construction is localized to the periplasm, but the Aida-I autotrans<porter can’t encroach to the external membrane but still conserve its proteolytic activity to release the passenger protein, which explains that the molecular weight corresponds to a free-sfGFP.

-Our construction is not taken in charge by the Sec Pathway, and so it isn’t directed to the periplasm and takes its conformation in the cytoplasm; and due to the mutation that we forgot to integrate into aida-I sequence, the passenger protein is cleaved inside of the cell.

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