Part:BBa_K314110

f1 origin

Phage origin recognized by M13. Used to make SS DNA when infected with M13k07 helper phage.

Usage and Biology

Origin of replication of M13 phages.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 126
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

Figure 1. Single stranded DNA harvested from phage and run on a gel. The higher bands are phage genomic DNA while the lower bands at roughly 2-3kb correspond to the expected plasmid size.

[[Image:|https://static.igem.org/mediawiki/2015/d/d6/Gel_phage_kinetic.pngthumb%7Cright%7C300px%7CFigure 2. 0.8% agarose gel electrophoresis. Loadad as follows: Lanes 1)Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) from NEB (10 ul), 2, 3,4) ssDNA 1hr (8 ul + 4 ul Loading buffer), 6,7,8,9) ssDNA 2hr (8 ul + 4 ul Loading buffer), 10, 11, 12, 13) ssDNA 3 hr. (8 ul + 4 ul Loading buffer). ]]

The f1 origin was tested by comparing single strand DNA harvest using part of the [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD Kunkel mutagenesis] protocol. CJ236 cells were infected with M13K07 helper phage. The CJ236 cells varied in the present or absence of the f1 origin on the pSB1A3 or pSB3K3 plasmid. The SS DNA harvest was then run on a 50ml 1% agarose gel at 90V for 45minutes. As is clearly visible on the gel to the right single strand DNA of the plasmid is only made when the f1 origin is present.

TecCEM 2015 characterised this biobrick in order to prove the theory of recovering ssDNA for aim of our project.ssDNA harvesting was done 1, 2, and 3 hours after M13 helper phage infection. This was verified by an agarose gel electrophoresis as shown in Figure 1, and D.O. (260 nm) was measured for each sample in order to determine the amount of ssDNA.The latter can be observed on Figure 2, where the correlation factor and the lineal regression equation are included. This is considered an indication of the increasing ssDNA plasmid result of the infection.

Figure 2. 0.8% agarose gel electrophoresis. Loadad as follows: Lanes 1)Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) from NEB (10 ul), 2, 3,4) ssDNA 1hr (8 ul + 4 ul Loading buffer), 6,7,8,9) ssDNA 2hr (8 ul + 4 ul Loading buffer), 10, 11, 12, 13) ssDNA 3 hr. (8 ul + 4 ul Loading buffer).

The quantification analysis can be observed on Figure 2, where the correlation factor and the lineal regression equation are included. The yield analysis could be further modelled with more samples through time.

Figure 2. Graph showing yield of ssDNA vs time. It is also shown the linear regression equation and the correlation factor.