Difference between revisions of "Part:BBa K1445000"

Latest revision as of 12:16, 17 October 2014

M13 Ori: Packaging signal for M13 phage

The M13 origin of replication is a non-coding sequence that facilitates the uptake of a plasmid as a single-stranded phagemid into the M13 or Fd phage. This part does not code for phage coat proteins; therefore cannot produce phage.

Usage and Biology

The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage. When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. To verify functionality, its packaging efficiency was compared to that of amilCP. The amilCP sequence is roughly equal in length to the M13ori and both parts were cloned onto the pSB1C3 backbone; therefore, the nucleotide sequence is the only distinguishing factor. The helper phagemid M13K07 (a plasmid containing M13 phage structural proteins and a weakened packaging signal) made phage in host cells containing pSB1C3-M13ori or pSB1C3-amilCP phagemids. ER2738 E. coli. were infected by phage from the M13ori or amilCP sample to assess their respective packaging efficiencies.

Figure 1: Conjugated BW23115 E. coli infected with recombinant phage A) pSB1C3-M13ori or B) pSB1C3-amilCP phagemids on LB agar with 170 ug/mL chloramphenicol.

The substantial growth for the M13ori sample in Figure 1A) confirms that pSB1C3-M13ori was successfully packaged and delivered. Conversely, the amilCP sample in Figure 1B) showed no growth indicating that none of the phage in this sample packaged pSB1C3-amilCP. The M13ori is necessary and sufficient for uptake into the M13 phage.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ===Usage and Biology===
-The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage.  When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. To verify functionality, its packaging efficiency was compared to that of amilCP. The amilCP sequence is roughly equal in length to the M13ori and both parts were cloned onto the pSB1C3 backbone; therefore, the nucleotide sequence is the only distinguishing factor. The helper phagemid M13K07 (a plasmid containing M13 phage structural proteins and a weakened packaging signal) made phage in host cells containing pSB1C3-M13ori or pSB1C3-amilCP phagemids. ER2738 E. coli. were infected by phage from the M13ori or amilCP sample to assess their respective packaging efficiencies.
+The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage.  When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. To verify functionality, its packaging efficiency was compared to that of amilCP. The amilCP sequence is roughly equal in length to the M13ori and both parts were cloned onto the pSB1C3 backbone; therefore, the nucleotide sequence is the only distinguishing factor. The helper phagemid M13K07 (a plasmid containing M13 phage structural proteins and a weakened packaging signal) made phage in host cells containing pSB1C3-M13ori or pSB1C3-amilCP phagemids. ER2738 ''E. coli''. were infected by phage from the M13ori or amilCP sample to assess their respective packaging efficiencies.
-[[File:Boulder_M13ori_FIGURE_1.png|600px|thumb|none|Figure 1: Conjugated BW23115 E. coli infected with recombinant phage  A) pSB1C3-M13ori or B) pSB1C3-amilCP phagemids on LB agar with 170 ug/mL chloramphenicol.]]
+[[File:Boulder_M13ori_FIGURE_1.png|600px|thumb|none|Figure 1: Conjugated BW23115 ''E. coli'' infected with recombinant phage  A) pSB1C3-M13ori or B) pSB1C3-amilCP phagemids on LB agar with 170 ug/mL chloramphenicol.]]
 The substantial growth for the M13ori sample in Figure 1A) confirms that pSB1C3-M13ori was successfully packaged and delivered. Conversely, the amilCP sample in Figure 1B) showed no growth indicating that none of the phage in this sample packaged pSB1C3-amilCP.  The M13ori is necessary and sufficient for uptake into the M13 phage.