Part:BBa_K4968006
RecomSwapNeo R/Kan R
RecomSwapNeo R/Kan R (BBa_K4968006) includes the kanamycin, Neomycin, and two homologous fragments which are settled up and downstream of the gene CsgA and CsgB. This part which is the improvement of Low to medium copy Lambda Red recombineering compatible plasmid (BBa_K592202) will be amplified by PCR and inserted into the MC4100 and BL21(DE3) strains, which can be used for collaborating with recombinase from plasmid pKD46.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 67
Illegal XbaI site found at 1459
Illegal PstI site found at 636 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 636
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 423
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 67
Illegal XbaI site found at 1459
Illegal PstI site found at 636 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 67
Illegal XbaI site found at 1459
Illegal PstI site found at 636
Illegal NgoMIV site found at 1087
Illegal NgoMIV site found at 1370 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 936
Illegal SapI.rc site found at 1146
Usage & biology
This linear fragment is used for a method called Lambda Red, which employs homologous recombination to knock out genes in the bacterial genome. It has been known for a long time that some bacteriophages possess their own homologous recombination systems (Smith, 1988). Several studies have indicated that when linear fragments are used, the recombination rate of the Lambda Red system is significantly enhanced (C. Murphy, 1998).
This recombinant fragment is designed and customized to include kanamycin, neomycin (both are part of the pKD4 plasmid gene), and two homologous fragments of the bacterial genome in strains MC4100 and BL21(DE3). These two homologous fragments are located approximately 50 base pairs upstream and downstream of the CsgA and CsgB genes (A. Sawitzke et al., 2013). This recombinant fragment functions in conjunction with the recombinase from the pKD46 plasmid (A. Datsenko et al., 2000).
In comparison to the original part BBa_K592202, we retained the antibiotic resistance genes within it and added the NeoR marker, which is originally capable of expressing neomycin. This addition allows this part to have the ability to function in eukaryotic organisms, facilitating understanding for future users. Not only does this antibiotic-resistant gene function in prokaryotes, but it can also operate in eukaryotes. Additionally, we replaced the original part's circular structure with a linear fragment, which makes Lambda Red homologous recombination more convenient and eliminates many unnecessary steps. Furthermore, we added homologous arms for CsgA and CsgB genes before and after the genes, facilitating specific knockout of these genes in the future.
After gene synthesis, as shown in Figure 1, we conducted PCR and agarose gel electrophoresis to confirm the correct construction of this part.
Figure 1 | The agarose gel electrophoretogram of the PCR product of the pUC57-kan-RecomSwapNeo R/Kan R plasmids. The product is the fragment used for homologous recombination which is 1528bp. The brightest band is near 1500bp from the electrophoretogram, which is the RecomSwapNeo R/Kan R fragment. 
After the substantial amplification of the linear fragment, RecomSwapNeo R/KanR, the pKD46 plasmid is essential to provide the required recombinase for homologous recombination. Notably, pKD46 is temperature-sensitive and will be lost at temperatures exceeding 37°C, aiding its elimination once successful recombination has occurred (Doublet et al., 2008). Upon insertion of the recombinant fragment and pKD46 into the target bacterial strains MC4100 and BL21(DE3), arabinose induction is necessary to activate recombinase expression.
In our specific procedure, we chose to employ LB plates containing both ampicillin and kanamycin for the selection of recombinant strains. Subsequently, we isolated the colonies and incubated them in LB broth containing only kanamycin at 37°C, effectively removing the pKD46 plasmid. This step serves as an initial screening, as illustrated in Figure 2. Additionally, DNA agarose gel electrophoresis is employed to confirm the precise size of the fragment between the two homologous arms, as demonstrated in Figure 3.
Figure 2 | The strain growth on plates after Lambda red homologous recombination. After lambda red homologous recombination by electroporation, the strain is grown on LB plates containing kanamycin, and the strain with successful knockout will carry kanamycin resistance and grow normally, while the failed knockout will not grow. Strains circled in red are used as subsequent colony PCR steps for further validation. Figure 3 | The agarose gel electrophoresis of the DNA fragment in the genome from BL21(DE3). Sample 1, sample 2, and sample 3 show the strips are 1 kb. Sample 5 has the highlight strip which is near 1.5kb. The length of the highlight strips is the same as the recombinant fragment which is 1528bp. This result means the CsgB and CsgA have been knocked out. Figure 4 | The agarose gel electrophoresis of the DNA fragment in the genome from MC4100. Sample 1, sample 2, and sample 3 show the strips are 1 kb. Samples 5 and 6 have the highlight strip which are near 1.5kb. The length of the highlight strips is the same as the recombinant fragment which is 1528bp. This result means the CsgB and CsgA have been knocked out.
RecomSwapNeo R/Kan R (BBa_K4968006) offers promising future applications for precise gene knockout and genetic manipulation in a wide range of organisms, including prokaryotes and eukaryotes. Its potential lies in facilitating targeted gene removal of Lambda red method while allowing for strain selection and survival through antibiotic resistance markers. This versatility makes it a valuable tool for advancing genetic research and biotechnological applications, including gene function studies, pathway engineering, and synthetic biology projects in various host systems.
Source
The RecomSwapKanR fragments are from pKD4. The homologous fragments up and downstream of the CsgA and CsgB in the genome are from NCBI.
References
Datsenko, K.A. and Wanner, B.L. (2000) ‘One-step inactivation of chromosomal genesin Escherichia coli K-12 using PCR products’ BIOLOGICAL SCIENCES 97(12)6640-6645. Available at: https://doi.org/10.1073/pnas.120163297
Doublet, B. et al. (2008) ‘Antibiotic marker modifications of 入 Redand FLP helper plasmids, pKD46 andpCP20, for inactivation of chromosomalgenes using PCR products in multidrugresistant strains’ J Microbiol Methods 75(2):359-61. Available at: https://doi.org/10.1016/j.mimet.2008.06.010
Murphy, K. C. (1998) ‘Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli’ Journal of bacteriology, 180(8), 2063–2071. Available at: https://doi.org/10.1128/JB.180.8.2063-2071.1998
Sawitzke, J.A. et al. (2013) ‘Chapter Ten - Recombineering: HighlyEfficient in vivo Genetic Engineering usingSingle-strand Oligos’ Methods in Enzymology 533,157-177. Available at: https://doi.org/10.1016/B978-0-12-420067-8.00010-6
Smith, G. R. (1988) ‘Homologous recombination in procaryotes’ Microbiological reviews, 52(1), 1–28. Available at: https://doi.org/10.1128/mr.52.1.1-28.1988
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