Translational_Unit
f1 ori

Part:BBa_K314110

Designed by: IGEM 2010 Team Washington   Group: iGEM10_Washington   (2010-10-08)

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:|http://2015.igem.org/wiki/images/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 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.

Gel_phage_kinetic.png

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.

OD_phage_TecCEM.png

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



Functional Parameters: Austin_UTexas

BBa_K314110 parameters

Burden Imposed by this Part:

Burden Value: -3.5 ± 5.9%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

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Parameters
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