Part:BBa_K4888003
SpaC, mucus binding protein, D2 domain
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, only D2 was retained using HiFi method. [2]
To see the construct with D1 and D2, look at BBa_K4888004.
Truncation of SpaC to D2
Method
In fact, due to the D2 domain sequence being nestled between two segments of the D1 sequence, a multi-phase approach is needed, culminating in Hifi cloning.
The cloning of D2 alone via Hifi is illustrated below :
Schematic of the Hifi cloning of 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.
Following an initial PCR, two fragments are retained: the backbone without D1 and the stalk region, and a separate fragment containing the D2 sequence. Subsequently, a second PCR is conducted using the D2 fragment, with the addition of homology arms to its sequence. Finally, the backbone and D2 fragments are merged using the Hifi method.
Experiment
The cloning process begins with the amplification of essential fragments through a PCR reaction.
Both the D2 fragment intended for cloning and its corresponding backbone were effectively amplified, revealing bands at the anticipated sizes of 765 bp and 4315 bp, respectively.
Agarose Gel Electrophoresis of PCR Amplification for the D2 and backbone Fragment
To facilitate a HiFi cloning reaction and merge these two fragments, the introduction of homology arms (HA) to the D2 fragment was essential. This was achieved by using primers that carried the desired HA sequences. During the second round of PCR, we successfully generated a visible fragment at the expected size of 765 bp, as demonstrated in our agarose gel electrophoresis results.
Agarose Gel Electrophoresis of PCR Amplification for the D2 Fragment with introduced Homology Arms (HA)
The D2 fragment was successfully incorporated into the backbone, and the bacteria transformation was successful.
Sequencing
After the cloning process, sequencing has been performed to validate the precision of the construct.
Below you can find the sequencing outcomes for D2, all of which were accurate. Some discrepancies at the fragment boundaries were detected, which is a common occurrence.
MicroSynth Sequencing results for the plasmid containing the D2-construct after HiFi cloning. Red lines indicate errors
The truncation is successful.
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] NEBuilder® HiFi DNA Assembly
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 451
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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