Fig 1.
A timeline of molecular cloning techniques.
The available cloning techniques can be classified into four types: (A) restriction endonucleases, (B) ligation-dependent staining, (C) in vitro assembly, (D) in vivo assembly.
Fig 2.
A schematic diagram illustrating the high-throughput construct platform based on the high-throughput FastCloning method.
The second-generation high-throughput prokaryotic expression vectors share the same sequence elements, including a ccdB gene expression core box (red box), upstream box for primer HighR binding (blue box), and downstream box for primer HighF binding (green box). The same pair of HighF and HighR primers can linearize all 12 pSDB1–B12 vectors via PCR, whereas primers for amplifying insert fragments share a homologous overlap for the upstream or downstream box, which facilitates high-throughput cloning of a single gene in multiple vectors. The insert fragment was mixed with the linearized vector, and the recombinant plasmid was used to transform Escherichia coli DH5α competent cells. In the recombinant plasmid, the insert fragment replaced the ccdB box, thereby facilitating DH5α cell survival, whereas ccdB protein expression from non-recombinant plasmids was lethal to DH5α. P in the figure represents promoter. Apr: Ampicillin resistance gene. Kar: Kanamycin resistance gene.
Table 1.
pFastB1~B12 original vector information.
Fig 3.
Recombination validation and protein expression analysis for the high-throughput FastCloning method.
(A) Recombinant plasmids containing the Mqo gene were validated by PCR, using the same primers used to amplify the insert. Validation of the ThrS and IleS recombinant plasmids is shown in (C) and ©, respectively. (B) The Mqo recombinant plasmids were used to transform the Escherichia coli BL21 (DE3) strain, and recombinant protein expression was analyzed using SDS-PAGE. Fusion proteins of the expected size are denoted by black triangles. The analysis of ThrS and IleS protein expression is shown in (D) and (F), respectively.
Fig 4.
A schematic diagram illustrating the multiple gene co-expression system based on the high-throughput FastCloning method.
Multiple gene co-expression systems were constructed using a stepwise approach. Step 1: Gene1 is inserted into a high-throughput vector. Step 2: The recombinant plasmid G1-Vector is linearized using the HighF and R1 primer pair. A 25-bp linker sequence is added to the linearized G1-vector fragment using the R1 primer. G2-HF shares a homologous 18-bp (denoted in yellow) overlapping with the R1 primer. Step 3: The G1-L-G2-Vector is linearized using the HighF and R2 primer pair, followed by the insertion of Gene3. Step 4: The G1-L-G2L-G3-Vector is linearized using the HighF and R3 primer pair, followed by the insertion of Gene4. All homologous sequences are indicated by the same color.
Fig 5.
Co-expression and co-purification of protein complexes based on the multiple gene co-expression system.
(A) Co-expression of the Trim6-Trim61 complex with two subunits of the Trim6-Trim61-pSDB1 vector. The Trim6 protein fused to a 6×His tag was harvested using Ni-NTA resin, whereas the untagged Trim6 protein bound tightly with the Trim6 protein forming a complex, which was eluted with imidazole buffer. (B) Co-expression of the Trim6-Trim61 complex in the Trim6-Trim61-pSDB7 vector. Trim61 did not fuse to a tag but formed a complex with Trim6 fused to a GST tag. The complex was purified by glutathione affinity chromatography. (C) Co-expression of the NuA4 complex comprising four subunits. Eas1 was expressed by fusing to a 6×His tag, and formed the NuA4 complex with the Epl1, Yng2, and Eaf6 subunits. This complex was then eluted with imidazole buffer. A contaminant protein is denoted by a black star. (D) Co-expression of the Lsm7-5-6-8-4-3-2 snRNPs complex comprising seven subunits in the pSDB1 vector. Lsm7 fused to a 6×His tag was co-eluted with other U6 snRNPs in a heptad protein complex. All target proteins are indicated by black triangles or labeled with the respective protein names.
Fig 6.
A comparison of high-throughput FastCloning (HTFC) with current optimal protocols for different cloning procedure.
(A) The experimental workflow for traditional restriction cloning procedure. (B) The experimental workflow for in-fusion cloning procedure. (C) The experimental workflow for high-throughput FastCloning procedure.