Mutants of bacteriophage lambda integrase
A technology of integrating enzymes and amino acids, applied in phages, viruses/phages, enzymes, etc., can solve problems such as difficult to transform cell lines
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Embodiment 1
[0186] Example 1: Rapid E. coli Chromosomal Integration
[0187] This example follows figure 2 method described in . C3INT-HIS-PET22b(+) was amplified with petF2 (SEQ ID NO: 10) and petR (SEQ ID NO: 11), followed by intramolecular ligation of the PCR products to generate the C3INT-HIS minicircle. attP-PET22b(+) was amplified with attP-F (SEQ ID NO:12) and attPSOE-R (SEQ ID NO:13), while TEM1prom-F (SEQ ID NO:14) and TEM1promR (SEQ ID NO: 15) Amplification of PET22b(+), which produces PCR products encoding attP and ampicillin resistance genes, respectively. Splicing Overlap Extension PCR (SOE-PCR) was performed using these two PCR products using attP-F (SEQ ID NO: 12) and TEM1prom-R (SEQ ID NO: 15). The PCR products were subsequently ligated intramolecularly to generate the attP-TEM1 minicircle. 100 ng of C3INT-HIS minicircle and 100 ng attP-TEM1 minicircle were combined and electroporated into 25 μL of electrocompetent TG1 cells. The cells were allowed to recover for 1 h...
Embodiment 2
[0188] Example 2: Recombination activity of parental and integrase variants C2 and C3
[0189] This example demonstrates the recombination activity of the parental Int-h / 218 and selected mutants (C2, C3 and their indicated variants). Figure 4A Depicted are results from in vitro intramolecular recombination reactions using integrase produced by in vitro transcription / translation. The plasmid encoding the corresponding integrase (Int-h / 218, C2, C3 or its variants) was amplified using primers IntRBS-F and INTstop-R, and the PCR product was reamplified with primers Univeral and INTstop-R to obtain in vitro Integrase amplicon of the T7 promoter and ribosome binding site required for transcription-translation (IVT). use The in vitro protein synthesis kit expressed 20 ng of each integrase amplicon in a total volume of 9 μL for 1 hour at 30°C. Then by adding attB / attP sites, attPH / attH sites or attH4x / attP4x sites ( figure 1 ) of 10 ng of plasmid substrate was added to a total v...
Embodiment 3
[0194] Example 3: Cell culture conditions, transfection procedure and selection of puromycin resistant recombinants for endogenous attH4x targeting in HT1080 cells
[0195] For endogenous targeting in the HT1080 cell line, the day before transfection, mix 3x10 6 Dulbecco's modified Eagle's medium [DMEM (Life technologies)] supplemented with 10% FBS, 1% L-glutamine, and 100 units / mL of penicillin and streptamine in each 10 cm cell culture dish. Mycin], 70-90% confluency was obtained at the time of transfection. Transfection was performed using Lipofectamine 2000 reagent (Life technologies). By combining 5ng targeting vector (pPGKssPuro-attP4x(SEQ ID NO:29)) and 100ng integrase expression plasmid (pCMVssKZ-IntC3-CNLS(SEQ ID NO:30)) diluted in 75μl Opti-MEM medium with the Plasmid DNA-lipid complexes were prepared by mixing 2.5 μl Lipofectamine 2000 reagent diluted in 75 μl Opti-MEM medium (Life technologies) and incubating at room temperature for 20 minutes. The transfection ...
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