Part:BBa_K1617000

Modular Flagellin - FliC_MCS

Flagellin is the filament forming component of bacterial flagellas. The up to 20 µm long filaments are formed by the self-assembly of flagellin. Flagellin proteins consist of four domains (D0-D3) and have a size of about 20-30kDa. While the D0 and D1 domains are well conserved, the D2 and D3 domains are highly variable in sequence and length [1]. Here, we created an modular flagellin with an easily replacable D3 region. A multi-cloning side flanking the D3 domain allows the design of costume-made flagellins by being compatible to iGEM fusion standards such as RFC25 and RFC21. You can pick any RFC25 compatible BioBrick from the registry and test its functionality immidiatally via an easy motility assay. This system enables the creation of a three-dimensional reactive nanostructure that has an increased specific surface with highly catalytic activity. With this method produced functionalized flagellas can be easily harvested via shearing stress following an easy protocol. Flaggelas are self-assembling systems. An temperature increase up to 95 °C depolymerizes the flagella and disconnects the flagellin subunits. By cooling down, these subunits assemble back into fully formed flagella filaments. This means that various flagellin subunits with different molecular set-up can be mixed and used to form multifunctional nanostructures. The resulting flagella may be constructed consisting of various different active sites, which will enable the combination of multiple enzymatic steps in close proximity. FliC MCS is a monomer which is stacked helically forming the flagella. In this part we used the fliC from Escherichia coli str. K-12 substr. MG1655. As the natural self-assembly involves the transportation of fliC thorugh the filament there are steric limitations to the flagellin design. If flagellin-hybrids with a higher molecular mass then the wildtype flagellin (about 52 kDa ProtParam) problems may arise due to steric effects. In this case, we suggest using non-nanonical aminoacids for in-vitro bioconjugation via click reaction. You can read more about that in our wiki or in our D3 Biobrick description.

Figure 1 Video showing the self-assembly of the bacterial flagella. FliC is the main component which makes up most of the filament. Source: https://youtu.be/GnNCaBXL7LY Figure 2 In the modular flagellin the not conserved D3 domain was replaced with a polylinker coding a multi-cloning site. This part may be used to introduce any standard RFC25 part into the filament forming flagellin.

Usage and Biology

You can use the reporters for measuring naringenin concentrations in your samples. Depending on which fluorophor you want to detect, you can use one of three biosensors:

A: with CFP response use BBa_K1497022 B: with mKate response use BBa_K1497021 C: with no reporter BBa_K1497019 D: with GFP response use BBa_K1497020

Figure 2 E. coli Top10 with different naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample BBa_K1497019 without fluorophor. Right: On agar plate with 100 µM naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible.

You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick K1497019 and clone your parts of interest (without RBS!) behind the device.

Functional Parameters

The Biobrick BBa_K1497019 produces in E. coli B and K strains the FdeR Protein. The iGEM Team TU Darmstadt 2014 measured the fluorescense of GFP and mKate after the incubation with diffrent conentrations of naringenin. The results are shown in Figure 3.

Figure 3 Left: Characterization of BBa_K1497020. GFP fluorescence depends on the concentration of naringenin. We measured the GFP fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of GFP as well. Right: Characterization of BBa_K1497021. mKate (BBa_K1055000) fluorescence depends on the concentration of naringenin. We measured the mKate (BBa_K1055000) fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.

In vivo characterisation of the naringenin biosynthesis operon (BBa_K1497007)

iGEM TU Darmstadt 2014 reconstitute the naringenin biosynthesis in E. coli by construction of a operon polycistronic gene cluster (BBa_K1497007) under control of the strong T7 promoter (BBa_K1497017). They used the naringenin biosensor with GFP response K1497020 to characterize the naringenin biosynthesis operon in E. coli BL21(DE3). The result are shown in figure 4. The GFP fluorescene is only in the cells with the T7 naringenin operon visible and detectable. The team determined for this operon a naringenin production yield of 3 µmol naringenin per liter.

Figure 4 Left: Cell pellets with and without T7-Naringenin operon from E. coli BL21(DE3)-pSB1C3-fdeR-gfp. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence. Right: Measurement of the GFP fluorescence in theE. coli BL21(DE3)-pSB1C3-fdeR-gfp strain containing and not containing the T7-Naringenin operon.

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Design of a functional flaggelin variant which does not contain the variable d3 flagellin domain anymore but instead carries a polylinker region (MCS). This polylinker region is the product of an inserted multi cloning side in place of the endogeneous d3 coding sequence.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 628
  Illegal BamHI site found at 577
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 583
  Illegal AgeI site found at 634
  
• 1000
  COMPATIBLE WITH RFC[1000]