Part:BBa_K3972002

ARG1 (Acoustic Reporter Gene 1)

E. coli codon-optimized ARG1 is a collection of 11 proteins that form gas vesicles when expressed. The proteins involved are GvpA, GvpC, GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU. ARG1 consists of two repeats of GvpA and five repeats of GvpC. These two proteins derive from Aphanizomenon flos-aquae and form the main structure and scaffold for the gas vesicle by dimerizing to form a biconical shape. The other proteins derive from Bacillus Mageterium and aid the formation and stability of the gas vesicles by acting as chaperone proteins. [1,2]

Usage and Biology

ARG1 is a protein which is under the control of the IPTG inducible promotor T7 (figure 1). The ARG1 proteins form gas vesicles which can be measured with an ultrasound.

Characterization

Expression
This part is optimized for expression of ARG1 in E coli cells. This protein was successfully transfected into BL21(DE3) cells as can be seen in figure 2.

Figure 2. Agar plate with transfected ARG1 in BL21(DE3) cells.

For protein expression, a small culture and large culture were made (Figure 3 &4). The conditions used during these culturing experiments were based on literature. [1]

Figure 3. Small culture of ARG1 in BL21(DE3).

Figure 4. Large culture of ARG1 in BL21(DE3).

The expression of the ARG1 proteins was characterized using ultrasound and SDS-PAGE. After a few attempts with the ultrasound, the right settings and set-up was found (Design Cycle). The first ultrasound images made were with 3 different IPTG concentrations namely 1 mM, 0.01 mM and 0.1 uM (Figure 5 & 6). The gas vesicles were imaged before and after collapse and the differences between those images were calculated to remove the background signal. Furthermore, these samples were purified using a centrifuge and characterized on an SDS-page (Figure 7).

Error creating thumbnail: Invalid thumbnail parameters

Figure 5. Ultrasound images pre & post and difference between those images.

Figure 6. SDS-page.

As can be concluded from the SDS-PAGE, the right proteins were formed since the bands on the SDS-PAGE match the mass of the proteins. As can be seen on the ultrasound images, there is a gradient in the intensity on the photos which corresponds to the different concentrations inducers used.

For a larger spectrum of ultrasound signals, the experiment was repeated with more IPTG concentrations: 10 mM, 1 mM, 0.1 mM, 1 uM and 0 uM (Figure 8 & 9). The gas vesicles were imaged before and after collapse and the differences between those images were calculated to remove the background signal. Furthermore, these samples were purified using centrifuge and characterized on an SDS-page (Figure 10).

Figure 7. Ultrasound images pre & post and difference between pre and post.

File:T—TU Eindhoven-- .png Figure 8. SDS-page.

Since the previous ultrasound pictures showed a gradient which corresponded to the induction concentrations, the same results were expected with these concentration inducers. As can be seen on the images, no gradient is visible from the lower to the higher concentrations. After discussions with our supervisors and consulting the article again, it was discovered that the concentration bacteria in our samples was too low. [1] This explains the low amount of gas vesicles imaged on the ultrasound images. Furthermore, the high IPTG concentrations can also kill the cells, which also results in low amount of gas vesicles on our images.[3]

References

[1] Bourdeau, R., Lee-Gosselin, A., Lakshmanan, A. et al. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 553, 86–90. https://doi.org/10.1038/nature25021

[2] Pfeifer, F. (2012). Distribution, formation and regulation of gas vesicles. Nat Rev Microbiol 10, 705–715. https://doi.org/10.1038/nrmicro2834

[3] Dvorak, P., Chrast, L., Nikel, P. I., Fedr, R., Soucek, K., Sedlackova, M., Chaloupkova, R., de Lorenzo, V., Prokop, Z., & Damborsky, J. (2015). Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway. Microbial cell factories, 14, 201. https://doi.org/10.1186/s12934-015-0393-3

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 851
  Illegal EcoRI site found at 4004
  Illegal XbaI site found at 602
  Illegal PstI site found at 3861
  Illegal PstI site found at 3966
  Illegal PstI site found at 4344
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 851
  Illegal EcoRI site found at 4004
  Illegal NheI site found at 736
  Illegal PstI site found at 3861
  Illegal PstI site found at 3966
  Illegal PstI site found at 4344
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 851
  Illegal EcoRI site found at 4004
  Illegal BglII site found at 1355
  Illegal BamHI site found at 4760
  Illegal XhoI site found at 4448
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 851
  Illegal EcoRI site found at 4004
  Illegal XbaI site found at 602
  Illegal PstI site found at 3861
  Illegal PstI site found at 3966
  Illegal PstI site found at 4344
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 851
  Illegal EcoRI site found at 4004
  Illegal XbaI site found at 602
  Illegal PstI site found at 3861
  Illegal PstI site found at 3966
  Illegal PstI site found at 4344
  Illegal NgoMIV site found at 1806
  Illegal NgoMIV site found at 2208
  Illegal AgeI site found at 4727
  
• 1000
  COMPATIBLE WITH RFC[1000]