Difference between revisions of "Part:BBa K1218011:Experience"

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[[File:Boulder_Improvement_upon_pCas9_FIGURE_1.png|600px|thumb|none|Boulder_Improvement_upon_pCas9_FIGURE_1.png]]
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[[File:Boulder_Improvement_upon_pCas9_FIGURE_1.png|600px|thumb|none|Figure 1: Transformation results of neomycin resistant E. coli with Cas9 part having either A) non-targeting or B) targeting spacer sequence.]]
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[[File:Boulder_Improvement_upon_pCas9_FIGURE_2.png|300px|thumb|none|Boulder_Improvement_upon_pCas9_FIGURE_2.png]]
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There was a substantial decrease in growth between the non-targeting (1920 colonies) and the targeting sample (8 colonies) that must be accredited to the differences in spacer sequence.
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As can be seen in Figure 1: B, there is growth in the targeting sample. Sequencing showed that all eight colonies had deleted the spacer region and one or both of the adjacent repeats.
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Part BBa_K1218011 was further improved by the addition of the M13 packaging signal (BBa_K1445000) to form the composite part BBa_K1445001. This part was packaged into phage and introduced via infection to conjugated neomycin resistant bW23115 E. coli. Sample was plated on chloramphenicol to select for cells infected with the phage delivering the phagemid with the Cas9 part and M13 origin of replication.
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[[File:Boulder_Improvement_upon_pCas9_FIGURE_2.png|300px|thumb|none|Figure 2: Infection results of BBa_K1445001 on a pSB1C3 backbone, grown on LB agar with 170 ug/mL chloramphenicol.]]
  
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The growth in figure 2 demonstrates that, with the addition of the M13 origin of replication, BBa_K1218011 can be delivered to cells via recombinant M13 phage.
 
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Revision as of 03:25, 11 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1218011

User Reviews

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Figure 1: Transformation results of neomycin resistant E. coli with Cas9 part having either A) non-targeting or B) targeting spacer sequence.

There was a substantial decrease in growth between the non-targeting (1920 colonies) and the targeting sample (8 colonies) that must be accredited to the differences in spacer sequence. As can be seen in Figure 1: B, there is growth in the targeting sample. Sequencing showed that all eight colonies had deleted the spacer region and one or both of the adjacent repeats.


Part BBa_K1218011 was further improved by the addition of the M13 packaging signal (BBa_K1445000) to form the composite part BBa_K1445001. This part was packaged into phage and introduced via infection to conjugated neomycin resistant bW23115 E. coli. Sample was plated on chloramphenicol to select for cells infected with the phage delivering the phagemid with the Cas9 part and M13 origin of replication.

Figure 2: Infection results of BBa_K1445001 on a pSB1C3 backbone, grown on LB agar with 170 ug/mL chloramphenicol.

The growth in figure 2 demonstrates that, with the addition of the M13 origin of replication, BBa_K1218011 can be delivered to cells via recombinant M13 phage.

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