Difference between revisions of "Part:BBa K3033024"

Revision as of 03:24, 22 October 2019

BBa_K3033024: FtnA overexpression

Overview

Our third and last feature of our whole Self-Activating Nanoparticle E.coli organism (SANCE). This feature would give our modified E.coli the ability to be magnetized as a control system for our project; this would be achieved through the overexpression of the FtnA. The general overview of the whole feature is shown in Figure 1 below.

Figure 1: General overview of the FtnA overexpression mechanism

The precise spatial arrangement of cells has been one of the most important topics in synthetic biology, and this feature also plays important role in our project, SANCE. After the surface capture system is activated by self-secreting tyrosinase and capture nanoparticles by the adhesive fusion protein on its surface, the E.coli – nanoparticles complexes should be removed from the wastewater treatment system to complete its mission. Hence, we designed our E.coli to obtain the third feature of our project, the magnetization. With this feature, our E.coli could be magnetized and be removed away from wastewater treatment process when a magnetic field is applied as shown Figure 9.

We accomplished this feature by cloning and overexpressing FtnA, which encodes for a globular protein complex called ferritin, consists of 24 protein subunits to form a nanoshell to store iron molecules by various metal-protein interactions ("Ferritin protein nanocages—the story"). Therefore, with the overexpression of FtnA, more ferritin proteins can help E.coli to store more iron within cytosol. [4.2] We expect that the magnetization of E.coli could be achieved within 30 minutes to 1 hour in real time observation. Thus, the removal of E.coli – nanoparticles complexes in wastewater treatment could be done within short duration.

Protein expression test

Figure 2: Western blotting protein expression band signal results from different strains of E.coli

Figure 3: The bar chart represents the different strains and their relative protein expression levels

Different E.coli strain types can influence FtnA protein expression levels. To investigate which E.coli strain is the best strain for Ftna protein expression, we ran western blot with the extracted protein samples from 6 different kinds of bacteria strains (Figure 2). We used rpoB (DNA-directed RNA 9polymerase subunit beta) as an internal control for our western blot analysis. Band signal’s intensity from the membrane were analyzed using Image Studio. After being normalized against the internal control (rpoB), we can see that different protein expression levels could be observed in different bacteria strains and all BL-21 derivatives strains which includes Rosetta, Star, pLys and DE3 showed a much higher level of protein expression compared to wild-type BL-21 strain (WT) as well as Stbl3 and DH5α strains.

Based on the results above, by using BL-21(DE3) we then proceeded to investigate the relative protein expression of this part in different arabinose concentrations.

Figure 4: Western blot result containing our protein bands which we later analyzed

Figure 5: Bar chart to represent the relative expression level and arabinose concentration

To investigate whether concentrations of arabinose will affect FtnA protein expression level, we ran western blot with bacteria lysis sample from BL-21 (DE3) bacteria incubated with 6 different concentrations of arabinose: 0-13300uM (Figure 4). Band signal’s intensity from the membrane were analyzed using Image Studio. Normalized with internal control rpoB (which the protein size is 150kDa), we can see that FtnA protein expression level rises when the arabinose concentrations are increased (Figure 5). Therefore, it indicates that the concentrations of arabinose can affect the FtnA protein expression level in the same bacteria strain. We have used both of these results for the characterization of the part number BBa_K1679029 by OUC China 2015.

Functional magnetization tests

Figure 6: Bar chart to represent the relative expression level and arabinose concentration

After testing the protein expression level, we also want to know whether those expressed proteins are functional. A functional FtnA protein would increase the magnetization efficiency; we expected that after 1 hr, there would be significant migration of the bacteria toward the magnet. Therefore, we did a magnetization test: The pBAD24-FtnA construct was transformed respectively into the six different strains which are BL21 (Rosetta), BL-21 (Star), Stbl3, BL-21 (pLys), DH5 alpha and BL-21(DE3). Our negative control is the wild type BL-21 DE3 which is without the construct. Overnight cultures of 5 mL were made and after overnight culture they were all induced by 0.02% arabinose and cultured overnight. The next day, 1ml of 100mM ferric ammonium citrate for 24 hours was added. After incubation, we centrifuged the bacteria culture, resuspended it with autoclaved water and poured 2ml into the small dish. A round shape magnet was set under each small dish during the 1hour magnetization. In figure 6, we can see that the strains, which includes BL-21 Rosetta, BL-21 Star, BL-21 pLys and BL-21 DE3 showed a clear bacteria migration towards the shape of the magnet after 1 hour. Meanwhile, no significant change can be observed in the wild type BL-21 strain (DE3) as well as Stbl3 and DH5α strains. This functional test results shows consistent results with our protein expression test result in Figure 5, and it also can demonstrate that our FtnA overexpression system can work in these strains that we would recommend using in the future.

These results would be a valuable characterization contribution to the said part for future teams who wishes to use the part and express the protein under the araBAD promoter.

Sequence and Features