Part:BBa_K108005

FlgH

flagellar protein of basal-body outer-membrane L ring of E.coli

Contribution From CAU_China 2023

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Group: CAU_China, 2023 https://2023.igem.org/Team:CAU_China

Author: Huang yaohan Zhang xiyuan Sun qianhui

Summary:Verified that this basic Part can work in Escherichia coli and we added some experimental conditions and result images

Biology

The SpyCatcher-SpyTag system was developed by the Howarth laboratory based on the internal isopeptide bond of the CnaB2 domain of FbaB, a fibronectin-binding MSCRAMM and virulence factor of Streptococcus pyogenes^[1].

Improvement

SpyTag is a short peptide consisting of 13 amino acids. The aspartic acid side chain in SpyTag can form isopeptide bonds with the lysine side chain of SpyCatcher^[2]. In particular, the size of SpyTag is equivalent to many epitope tags, which can be produced as fusion proteins and can be applied in the direction of antigen delivery, modification of protein hydrogels, etc.</> <p>We attempted to display SpyTag and SpyCatcher on the surface of Escherichia coli BL21(DE3) respectively, using this system to achieve cross-linking between bacteria.

Using fluorescent proteins, we constructed a system for verifying cross-linking, in which the engineered bacteria introduced plasmids and genes as shown in the table below.

<thead> </thead> <tbody> </tbody>

	pET30a	pJUMP46-2A
A	SpyTag	sfGFP
B	SpyCatcher	mCherry
C	empty plasmid	sfGFP
D	empty plasmid	mCherry

Tab1. Plasmids andgenes induced into engineering bacteria.

We verify cross-linking in two ways: by measuring optical density and microscopy.

Due to the cross-linking between bacteria, the buoyancy increases, and after standing for a period of time, fewer bacteria settle down, and the remaining rate of bacteria is greater.

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Fig1. Quantitative verification of adherence of bacteria.

Fluorescence microscopy and confocal microscopy were used to verify the cross-linking, and four groups of experiments were set up, namely the control group (AD, BC, CD) and the experimental group (AB). The observation results were shown in the figures below.

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Fig2. Observation of bacterial adhesion by laser microscopy Observation of bacterial adhesion by laser microscopy were observed under a laser microscope (1000×).

It can be seen from the above figure that the bacteria in the experimental group have obvious aggregation phenomenon, and the fluorescence in them can be seen that the aggregated bacteria express SpyTag and SpyCatcher respectively, which shows that the system can work.

References of CAU_China

[1] Hatlem, Daniel et al. “Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.” International journal of molecular sciences vol. 20,9 (2019): 1-10. doi:10.3390/ijms20092129</p>

[2] Kozlowski, Mark T et al. “Genetically Programmable Microbial Assembly.” ACS synthetic biology vol. 10,6 (2021): 1351-1359. doi:10.1021/acssynbio.0c00616</p>

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 114
  
• 1000
  COMPATIBLE WITH RFC[1000]