Part:BBa_K3972002

ARG1 (Acoustic Reporter Gene 1)

E. coli codon-optimized ARG1 is a collection of 12 proteins that together 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 four repeats of GvpC. These two proteins are derived from the cyanobacteria species Aphanizomenon flos-aquae and form the fundamental structure and scaffold for the gas vesicles by dimerizing to a biconical shape. The other proteins derive from Gram-positive bacteria Bacillus Megaterium and assist the formation and stability of the gas vesicles by acting as chaperone proteins [1,2].

Usage and Biology

ARG1 is a protein that is under control of the IPTG inducible T7 promoter (Figure 1). The ARG1 proteins form hollow nanostructures that contain different kinds of natural dissolved gasses. These protein vesicles produce contrast in ultrasound images, due to the ability of the proteins to scatter the incoming sound waves of the ultrasound transducer. Using acoustic pulses with an amplitude above the critical collapse pressure, the gas vesicles can be snapped [1]. The images can be taken before and after the collapse of the gas vesicles and can be substracted, to obtain clear images of the amount of gas vesicles present in the bacteria. The gas within the GV is unable to escape the protein shell in the timeframe the GV collapses, resulting in a compression of gas that prevents the complete destruction of the GV. GVs are highly effective acoustic reporters as a result of their nondestructive buckling, and nonlinear signal, which enables the subtraction of background noise for higher spatiotemporal resolution. The nonlinear properties of GVs have been utilized to improve imaging methods beyond linear arrays. Maresca et. al shows that the amplitude modulation (AM) sequence can be used to find the backscattering created by two consecutive transmissions of different amplitudes above and below the collapse pressure. This method shows a higher resolution in vivo and in vitro, albeit in vivo produced images suffer from image artifacts created by the nonlinear signals [4].

Figure 1. The mechanism of ARG1

Characterization

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

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

As can be seen in figure 3 &4, for ARG1 protein expression a small culture and a large culture were made. 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 equipment and SDS-PAGE. After a few attempts to optimize the ultrasound imaging, the right settings and set-up was established (Design Cycle). The first successful experiment to obtain ultrasound images was executed by inducing cultures with3 different IPTG concentrations, namely 1 mM, 0.01 mM and 0.1 µM (Figure 5). These induced cultures had to be anchored into a phantom to limit movement of the bacteria. The phantoms consisted of a mixture of PBS agar and the induced large cultures and were poured into petri dishes. The gas vesicles were imaged before and after collapse and these images were substracted to remove the background signal. Furthermore, these samples were purified using a centrifuge protocol and visualized on an SDS-page (figure 6).

Figure 5. Ultrasound images of phantoms containing large culture with BL21 (DE3) induced with different concentrations IPTG. The images were made with the transducer at an angle with respect to the phantom containing 15 mL of 1% agar agar in PBS mixed with 15 mL large culture. left) raw images before (pre) and after (post) running the collapse script (white signal is reflection of the ultrasound signal). right) processed difference between the pre and post collapse images. The white signal relates to the concentration gas vesicles.

Figure 6. SDS-Page of the centrifuge purified ARG proteins. On the left a Precision Plus ProteinTM All Blue Standards was used. Then there are three slots loaded with purified protein from wild type ARG large culture induced with 1000 µM, 10 µM and 0.1 µM IPTG respectively. Just as in the ultrasound a gradiënt is visible in intensity of a band around 30 kDa (red rectangle), suggesting successful control over the production of gas vesicle proteins.

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.

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

[4] “Nonlinear X-Wave Ultrasound Imaging of Acoustic Biomolecules.” Physical Review X, vol. 8, no. 4, 4 Oct. 2018, www.ncbi.nlm.nih.gov/pmc/articles/PMC8147876/#:~:text=The%20xAM%20method%20derives%20from%20counter-propagating%20wave%20interaction, https://doi.org/10.1103/physrevx.8.041002.

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]