Team:Bilkent-UNAMBG/Results

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RESULTS

Throughout the summer we performed different characterization experiments in order to verify and test the functionality of both our biofilm producing and penicillin binding csgA+PBP1b construct and iron storage protein bacterioferritin (BFR) producing construct. In these characterization experiments we have observed the successful biofilm formation. Producing biofilms was a breakthrough since it has been a challenging task in our project. Despite the fact that we could not manage to verify the penicillin binding property of our biofilm structure, we have some promising 3D protein interaction modellings that motivates us to keep working on penicillin binding part of our project. The characterization of penicillin binding peptide is still on progress.

Although we couldn’t get any data indicating the presence of magnetic properties from our magnetism assays, we have successfully verified the presence of bacterioferritin. Following the verification of BFR, we improved this part that has been submitted by Berlin 2014 iGEM team by conjugating the BFR part with sfGFP and visualized the sfGFP signal.

Penicillin Binding Peptide: PBP1b

We performed immunocytochemistry (ICC) assay in order to observe the expression of csgA+PBP1b. Working principle of immunocytochemistry assay is to provide bacterial cultures that are producing His-Tag containing proteins with His-Tag binding antibodies and visualize the cells under fluorescence microscopy. With primary antibodies, we bind the His-Tag that is expressed with the csgA that is secreted to the cell surface.  After primary antibodies, secondary antibodies are used in order to label the primary antibodies with fluorescence activity. Under fluorescence microscopy these cells could be visualized. We performed immunocytochemistry assay to visualize if our csgA has His-Tag that is also attached to PBP1b. In our trials we couldn’t get any signal from fluorescence microscopy, as it can be seen in Figure 1. Only signal we could get was the signal of dead cells. Since we have verified our sequence and we expect csgA+PBP1b conjugated protein to be expressed, we concluded that different possibilities could cause this result; such as BSA blocking the 3D structure of PBP1b which is a very short peptide so that primary antibodies couldn’t bind the His-Tag.

 

Figure 1: Immunocytochemistry assay results for induced and uninduced biofilm producing constructs.

After our first trial and failure we tried producing biofilms on YESCA plates that contain congo red that dyes the amyloid fibers which are produced in biofilms to hold the bacteria together. MG1655 is a wild type bacterial strain that is known for its overexpressed curli operon that cause bacteria to form biofilms. In this assay, we expect MG1655 control to have slightly less red color than our induced ΔcsgA cells having our construct. Also we expect MG1655 control to have more reddish color than our uninduced ΔcsgA cells. We streaked our bacteria onto YESCA plates with congo red and followed the biofilm formation procedure. After 3 days of incubation at 30 ºC, we couldn’t see a significant difference between our induced, uninduced and control plates. Photo of our plates after 3 days incubation can be seen in Figure 2. The reason why we couldn’t see a significant difference between our plates is that the amyloid formation might not be that high in the arabinose concentration that we have used and the incubation time might not be enough for formation of stronger amyloid fibers.

 

Figure 2: Congo red assay for our biofilm producing construct using YESCA plates.

After not being able to get significantly positive data from immunocytochemistry and congo red assays, we decided to perform crystal violet assay and scanning electron microscopy (SEM) imaging of our biofilm producing constructs. We induced our biofilm producing bacteria in 24-well plates with 0.2% L-arabinose. Crystal violet stains the outer membrane of cells. After producing biofilm structures in 24-well plate, we expect our cells to adhere to the surface of the plate and the addition of crystal violet would stain the cells that have adherence. After several washes all residuals except for our biofilm producing bacteria would be removed in the presence of biofilm formation that eventually cause cells to adhere to the surface. After staining with crystal violet and washing the wells, we observed no stained cells indicating the absence of adherence and biofilm formation in Figure 3. After getting no supporting data from crystal violet, we prepared SEM imaging samples and visualized the cells under scanning electron microscopy. There were three samples for SEM which were empty ΔcsgA cells containing no constructs, uninduced ΔcsgA cells, and ΔcsgA cells that were induced with 0.2% L-arabinose. As expected we observed no amyloid fibers in empty ΔcsgA cells and uninduced ΔcsgA cells. And in the induced sample we observed the fiber formation despite being in a minor level in Figure 4. Not having a positive result from crystal violet but observing the fiber formation under SEM lead us to using higher amounts of L-arabinose and increased induction period for our biofilm producing bacteria.

 

Figure 3: Preparation of crystal violet (CV), congo red, and SEM imaging samples and the crystal violet result.

 

Figure 4: Scanning electron microscopy (SEM) images of empty, uninduced and induced ΔcsgA cells.

For the second trial of crystal violet assay, our setup consisted of 8 different sample types. We used 5 colonies that are verified to be carrying csgA+PBP1b construct. A colony with the original csgA that have not been modified as a positive control. Empty ΔcsgA cells with no constructs. And a co-transformation colony carrying both the PBP1b and BFR constructs. We increased the induction period to 6 days and inducer amount to 0.4% and 0.8%. We did not get any results for the 0.2% arabinose induction for 3 days however almost all the samples gave results as expected in the second trial with 6 days with increased inducer. And we did not observe a significant difference between the 0.4% inducer and 0.8% inducer. All these have brought us to the conclusion that rather than the inducer amount the increment in induction period affected our results and caused better biofilm formation. 

 

Figure 5: Second trial of crystal violet assay with different inducer amounts and induction periods.

After observing successful induction of biofilm producing cells in SEM imaging and crystal violet assay, we wanted to demonstrate the expression of our csgA+PBP1b conjugated protein in Western Blot and SDS-PAGE. However we couldn’t get any expected results as it can be seen in Figure 6. The reason for this situation might be that csgA is not an easy protein to be used in these two methods. Before observing either in SDS or Western Blot, csgA must be monomerized. For csgA to be monomerized there is a specific protocol that is required. Since we know our construct is working properly, failure in these two methods is most probably caused by unsuccessful monomerization of csgA.

 

After observing successful induction of biofilm producing cells in SEM imaging and crystal violet assay, we wanted to demonstrate the expression of our csgA+PBP1b conjugated protein in Western Blot and SDS-PAGE. However we couldn’t get any expected results as it can be seen in Figure 6. The reason for this situation might be that csgA is not an easy protein to be used in these two methods. Before observing either in SDS or Western Blot, csgA must be monomerized. For csgA to be monomerized there is a specific protocol that is required. Since we know our construct is working properly, failure in these two methods is most probably caused by unsuccessful monomerization of csgA.

 

Figure 6: Western Blot and SDS-PAGE results for csgA+PBP1b.

Bacterioferritin: BFR

In order to visualize the expression of BFR protein that we are using in our construct, we added His-Tag to the end of our protein and performed Western Blot. In Figure 7, it can be seen that we got our desired band in Western Blot. This result is an indicator of we successfully cloned BFR part and our construct is expressing BFR.

 

Figure 7: Western Blot result for BFR.

In our project, we first used mCherry fluorescent protein to visualize the BFR producing bacteria. But in further applications, we thought that using sfGFP instead of mCherry would be more beneficial due to sfGFP’s fluorescent signal strength in comparison to mCherry. And we used Gibson Assembly to clone sfGFP into our BFR containing backbone. We verified our BFR and sfGFP producing construct by visualizing colonies under transilluminator and colony PCR. Transilluminator and colony PCR results can be seen in Figure 8.

 

Figure 8: ProD BFR sfGFP cloning plate with sfGFP signal and colony PCR for 6 chosen colonies.

 

In order to test the efficiency of the magnetic property of our BFR producing bacteria, we designed a magnetism assay. We incubated our cells with 1mM Fe2+ ions for 3 hours and put them on slides. Then we put our cells into electromagnet having high magnetism effect. We were expecting our cells to migrate through the edges of the slides and get closer to the magnet. After applying magnetic force for a while, we put our slides under fluorescence microscopy and visualized the amount of bacteria present at both sides of the slides. We have seen that our cells were alive after iron treatment and moving under the microscope, but there was no significant migration of cells. Figure 9 is the image we have seen under fluorescence microscopy.

Figure 9: Fluorescent microscopy result from colonies having sfGFP signal. 

Since we verified our BFR sequence and we get sfGFP signal, we are sure that BFR protein is produced in our construct. Even though we produce BFR, we couldn’t get to observe the magnetic functionality. This may be due to either insufficient strength of our magnets or a suppression of BFR production in our construct