Difference between revisions of "Part:BBa K4614105"
 
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<partinfo>BBa_K4614105 short</partinfo>
 
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T7-RBS-wza-SpyCatcheris composed of T7 promoter, RBS and T7 terminator from plasmid pET-30a(+), wza gene (BBa_K4614104), SpyCatcher gene (BBa_K4614103) and GS linker (BBa_K4614106).  
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T7-RBS-wza-spycatcher is composed of T7 promoter, RBS and T7 terminator from plasmid pET-30a(+), wza gene (BBa_K4614104), spycatcher gene (BBa_K4614103) and GS linker (BBa_K4614106).  
 
This composite part is designed to use protein wza to display the protein SpyCatcher at a relatively fixed position on the surface of bacteria to give it the ability to cross-link at specific locations on the bacterial surface.  
 
This composite part is designed to use protein wza to display the protein SpyCatcher at a relatively fixed position on the surface of bacteria to give it the ability to cross-link at specific locations on the bacterial surface.  
 
Since there is a lac operator sequence behind the T7 promoter of plasmid pET-30a(+), the display effect of SpyCatcher can be artificially controlled by the time and concentration of IPTG addition.
 
Since there is a lac operator sequence behind the T7 promoter of plasmid pET-30a(+), the display effect of SpyCatcher can be artificially controlled by the time and concentration of IPTG addition.
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===<span class='h3bb'>Sequence and Features</span>===
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<partinfo>BBa_K4614105 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4614105 parameters</partinfo>
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Latest revision as of 15:34, 12 October 2023


T7-RBS-Wza-SpyCatcher

T7-RBS-wza-spycatcher is composed of T7 promoter, RBS and T7 terminator from plasmid pET-30a(+), wza gene (BBa_K4614104), spycatcher gene (BBa_K4614103) and GS linker (BBa_K4614106). This composite part is designed to use protein wza to display the protein SpyCatcher at a relatively fixed position on the surface of bacteria to give it the ability to cross-link at specific locations on the bacterial surface. Since there is a lac operator sequence behind the T7 promoter of plasmid pET-30a(+), the display effect of SpyCatcher can be artificially controlled by the time and concentration of IPTG addition.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 704


Source

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].

Characterization

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.

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.

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.

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.

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

[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

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