Part:BBa_K4614102

T7-RBS-FlgH(SpyTag)

In this project, FlgH is derived from the genome of E. coli str. K12, and SpyTag and other components are synthesized by the company.

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].

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 157
  
• 1000
  COMPATIBLE WITH RFC[1000]

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

Table 1. Plasmids and genes 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.

Fig.1 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.

Fig.2 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.

Activity Analysis

Cross-linking

​ SpyCatcher can form isopeptide bonds with SpyTag, and we hope to achieve cross-linking between bacteria through this system.The Wza-SpyCatcher we created can realize the localized expression of SpyCatcher at the poles of Escherichia coli to form special microstructure.

Construction of SpyTag-SpyCatcher system and verification of cross-linking by OD₆₀₀ determination

​ To make the cross-linking more regular, SpyTag is fused with FlgH and SpyCatcher is fused with Wza, so that SpyTag will be displayed at the base of the flagella and SpyCatcher will be displayed at the poles of the bacteria.

​ Using the One Step Cloning II Kit (Vazyme Biotech, China) , we successfully constructed two gene circuits: T7-RBS-flgH-spyatg(BBa_K4614102) and T7-RBS-wza-spycatcher(BBa_K4614105), and transferred them into pET-30a(+) after sequencing.

Fig.3 Genetic circuit we used to test spytag and spycatcher. ((BBa_K4614102) and BBa_K4614105)

​ The groupings areas follows: the experimental group (SpyTag + SpyCatcher), control group 1 (SpyTag), and control group 2 (SpyCatcher). The combination remains the same as in table 2. We have increased the sampling times to 0 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes. We directly mix the bacterial cultures in cuvettes and let them stand.We measured OD₆₀₀ and calculated remaining.

Table 2. Combination

Fig. 4 The experimental group has the highest remaining at the end.

​ The experimental group maintains a consistently higher remaining value than the other groups after 1.5 hours, which aligns with our expectations. We confirmed cross-linking can happen, but we want to see more intuitive and cool results, and look forward to seeing where the cross-linking is located.

Laser microscopy

​ We hope to determine whether bacteria are cross-linked by direct observation.

Fig. 5 Genetic circuit for microscopy**((BBa_K4614102) , BBa_K4614105, BBa_K4614110, BBa_K4614111)**

​ We constructed the expression vector shown in the figure and introduced corresponding plasmids into the control group according to the table below. We named them A, B, C, D.

Table 3. Plasmids and genes induced into engineering bacteria.

​ Mix the two bacterial solutions in equal volumes according to the combination in Table 2 and shake well. Add 10 ul of the mixed solution onto the glass slide, immediately cover it with a coverslip, and observe under a laser microscope.

Table 4. Combination

​ We tried many times to find the best way to make samples. We tried to change the production method and use bacteria to gather themselves instead of manual mixing. At the same time, the culture time of bacteria is extended.

​ In the end we found that mixing the two bacterial solutions, waiting for 40 minutes, adding 10 microliters of water and waiting for 5 minutes was the most suitable. When smearing in this way, we could observe obvious cross-linked clumps in the experimental group, while none was observed in the control group.(Fig. 4,5)

Fig. 6 fluorescence microscopy (400×) In the experimental group (bacteria A and B), distinct clumps were observed, while in the other control group, the bacteria appeared to be randomly distributed.

Fig. 7 fluorescence microscopy (1000×). In the experimental group (bacteria A and B), distinct clumps were observed, while in the other control group, the bacteria appeared to be randomly distributed. More details are shown in the image above.

​ We tried to add different volume water to make samples of gradient concentration. As a result, as the dilution multiple increases, the cross-linking group block became smaller.(Fig. 6)

Fig. 8 As the dilution multiple increases, the cross-linking group block becomes smaller. 2x, 3x, and 4x dilution from left to right. The engineered bacteria used in these experiments are bacteria A and bacteria B in Table1.

​ After obtaining the best manufacturing sample conditions, we observed the samples through the laser scanning confocal microscope and obtained multiple 3D models of cross-linked structures (Fig. 7).

Fig. 9 3D models of cross-linked structures.

​ The display protein we designed is functional. Through direct observation, we found that the Wza did migrate to the pole of bacteria after filamentation as expected. As is shown in the white circle in Fig. 8, filamentated bacteria A (red, expressing Wza-SpyCatcher) is cross-linked with bacteria B (green, expressing FlgH-SpyTag) only at its two poles.

Fig. 10 Wza migrates to the pole of bacteria after filamentation. The left side shows the fluorescence microscope result, and the right side is the model diagram. The red rod represents the bacteria expressing SpyCatcher in filaments, and the green ball represents the bacteria expressing SpyTag.

Integration of Filamentation & Cross-linking module

Based on the cross-linking group of engineered bacteria ABCD, the specific information is shown in Table 1, we conducted the antibiotic filamentation experiment and tried to construct a new gene circuit to realize the cross-linking and filamentation binding at the molecular level.

Table 5. Plasmids and genes induced into engineering bacteria.

Cross-linked engineering bacteria antibiotic filamentation experiment

Based on literature review, we set up the following experimental group to use ampicillin to filamentation bacteria and simulate the possible cross-linking situation of filamentation bacteria after the expression of SulA gene to verify our conjusion:

Table 6. The process and group setup of cross-linked engineering bacteria antibiotic filamentation experiment

Laser microscope observation results are shown in Fig 1.

Fig. 11 The filamentated SpyCather could still be cross-linked to SpyTag under 400 × fluorescence microscope. It is obvious that the degree of red and green fluorescence overlap in the experimental group (SpyTag+SpyCatcher) is greater than that in the other three control groups.

In microscopic observations, we found that a lot of green fluorescence binds to the poles of the red coryneform, which means that Wza can indeed position SpyCatcher at the poles after bacterial filaments. Even more surprising, we observed that some bacteria crosslinked to form a special linear, square and lotus structure, which means that when our engineered bacteria express both crosslinking and filamentation modules at the same time, it is very likely to form a micro-structure with special properties.

Fig. 12 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - linear

Fig. 13 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - close to square shape

Fig. 14 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - lotus shape

These results demonstrate that there is a high probability that the materials formed by our engineered bacteria will have special microstructure and therefore special properties.We were extremely excited to get these results less than 24 hours before the Wiki freeze.

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