Part:BBa_K5061000
M13KO7 Helper Phage
This part is the M13KO7 Helper Phage from New England Biolabs (Cat#N0315S).
Usage and Biology
M13KO7 is a modified M13 phage that includes the origin of replication from p15A and a kanamycin resistance gene, both inserted into the M13 origin [Vieira & Messing, 1987]. The presence of these two elements allows it to replicate as a plasmid in E. coli cells.
M13KO7 is a helper phage suitable for producing single-stranded DNA from a phagemid carrying a wild-type M13 or f1 origin is present.
In the absence of a phagemid DNA, M13KO7 can replicate, pack and secrete single-stranded phage DNA
M13 Helper Phage derivatives were designed and used in the Phage-Assisted Continuous Evolution (PACE) [Esvelt et al., 2011] and variants PANCE [Roth et al., 2019], PRANCE [DeBenedictis et al., 2022], ...
Sequencing of this part revealed differences compared to the sequence available on New England Biolabs website; Some of this differences are present in the M13 helper phage VCSM13 (GenBank AY598820). A complete list is presented in Table 1
| Table 1. Sequence differences between this part, the wild-type M13 phage and the published sequences of M13KO7 and VCSM13 two commercially available M13 helper phages. Nucleotide numbers are based on this part sequence. | ||||||
|---|---|---|---|---|---|---|
| Region | Position | Nucleotide present in This Parts | Nucleotide present in NEB's M13KO7 helper phage | Nucleotide present in VCSM13 helper phage (GenBank AY598820) | Nucleotide present in M13 Phage (GenBank V00604) | Observations |
| M13 ori | 170 | G | G | T | G | intergenic region |
| gII | 340 | A | A | G | A | non synonymous mutation: A->G gII I51V |
| gVIII | 1908 | C | T | T | T | non synonymous mutation: T->C gVIII V6A |
| gIII | 2170 | G | G | G | A | non synonymous mutation: A->G gIII M1V |
| gIII | 2328 | C | T | C | T | synonymous mutation: gIII G53 |
| gIII | 3301 | G | A | G | A | non synonymous mutation: A->G gIII S378G |
| gIV | 5044 | A | T | A | T | synonymous mutation: gIV A78 |
| gIV | 5062 | A | T | A | T | synonymous mutation: gIV V84 |
| gIV | 5068 | T | A | T | A | synonymous mutation: gIV S86 |
| gIV | 5070-5071 | AC | TT | AC | TT | non synonymous mutations: TT->AC gIV I87N |
| gIV | 5080 | T | A | T | A | synonymous mutation: gIV I90 |
| gIV | 5338 | T | A | T | A | synonymous mutation: gIV A176 |
| gIV | 5377 | A | T | A | T | synonymous mutation: gIV S189 |
| gIV | 5965 | G | A | G | A | synonymous mutation: gIV Q385 |
| gIV | 6034 | T | C | T | C | synonymous mutation: gIV S408 |
| intergenic between ori15A and KanR in the AmpR résidus: | 7550 | / | insertion of 1 nucleotide: C | / | / | intergenic region |
| KanR RBS | 8550 | A | A | C | / | |
| KanR Promoter region | 8641 | G | G | A | / | |
| intergenic between KanR and in the AmpR rés | 8657 | C | ∆C | C | / | |
References
DeBenedictis EA, Chory EJ, Gretton DW, Wang B, Golas S, Esvelt KM. Systematic molecular evolution enables robust biomolecule discovery. Nat Methods. 2022 Jan;19(1):55-64.
Esvelt KM, Carlson JC, Liu DR. A system for the continuous directed evolution of biomolecules. Nature. 2011 Apr 28;472(7344):499-503.
Roth TB, Woolston BM, Stephanopoulos G, Liu DR. Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2. ACS Synth Biol. 2019 Apr 19;8(4):796-806.
Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3-11.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2811
Illegal XhoI site found at 8512 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1
Illegal NgoMIV site found at 6204
Illegal AgeI site found at 6458
Illegal AgeI site found at 6782 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6
Illegal BsaI.rc site found at 7487
| None |