Part:BBa_K4888004
SpaC, mucus binding protein D1/D2 domains
D1/D2 domain of SpaC mucus binding protein.
SpaC is a mucus-binding protein originating from a strain of Lactobacillus. For more information about SpaC BBa_K4888002
Recognizing that SpaC was too extensive for transport on the bacterial surface, we embarked on a strategy to reduce its size. This task involved the application of two distinct cloning techniques: Hifi and KLD. These methodologies allowed us to excise a portion of the SpaC sequence that wasn't necessary for mucus binding.
In their research, Kant, Abhiruchi, et al.5 [1] meticulously characterized the complete SpaC protein structure, identifying three domains, namely D3, D4, and D5, comprising the stalk region, while the D1 and D2 domains constituted the binding region.
In this design, D1 and D2 were preserved using the KLD method. [2]
To see the design containing D2 only, look at BBa_K4888003.
Truncation of SpaC to D1/D2
Method
The schematic depicting the cloning of D1 and D2 using KLD is presented below:
Schematic of the KLD cloning of D1 and D2
The initial construct is shown, encompassing SpaC with all its domains (comprising the stalk and binding region), the fused GFP nanobody, and flag tag. After KLD cloning, the stalk region is eliminated, leaving everything else intact in the construct.
Experiment
PCR was used to amplify the vector fragment housing the D1D2 construct, yielding a fragment of the anticipated size, which was 8620 base pairs as shown below. Following this, KLD cloning reaction was performed to ligate the ends of the fragment, resulting in the successful transformation of the plasmid.
Agarose Gel Electrophoresis of PCR Amplification for the vector Fragment for D1D2
Following this, bacterium were re-transformed with these new construct.
Sequencing
After the cloning process, sequencing has been performed to validate the precision of the construct.
In the figures below, the sequencing outcomes for D1D2 were illustrated. It was revealed in the sequencing data that a nucleotide was missing in the stop codon, resulting in an additional 10 amino acids at the C-terminal of our new construct. Some discrepancies at the fragment boundaries were detected, which is a common occurrence.
MicroSynth Sequencing results for the plasmid containing the D1D2-construct after KLD cloning. Red lines indicate errors.
Zoom on nucleotide loss in MicroSynth Sequencing results for the plasmid containing the D1D2-construct after KLD cloning. Red lines indicate errors.
The truncation is successful, despite the little nucleotide addition.
References
[1] Kant, Abhiruchi, et al. Crystal structure of lactobacillar SpaC reveals an atypical five-domain pilus tip adhesin: Exposing its substrate-binding and assembly in SpaCBA pili. Structural Biology 211.3 (2020): 107571
[2] KLD Enzyme Mix
https://www.neb.com/en/products/m0554-kld-enzyme-mix#Product%20Information
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 742
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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