Part:BBa_K2325104
N20-sgRNA function device in pTarget
The BBa_K2325104 is a construct contains N20 sequence selected from the genome of T7 phage, with a constitutive promoter, which is constructed in the CRISPR/Cas system to make the E.coli be able to confer resistance to T7 phage.
Background
The N20 is selected from the genome of T7 phage, which is responsible for the synthesis of the DNA packaging protein during phage infection. The 20-bp complementary region (N20) with the requisite PAM (NGG) matching genomic loci of interest was programmed directly into a heterologously expressed CRISPR array, and fused crRNA and tracrRNA as a single synthetic guide RNA (sgRNA) transcript obviated the need for processing the transcribed CRISPR array (pre-crRNA) into individual crRNA components.
Gel analysis
Usage and Biology
The pTarget-N20 processing this device is co-transfected with pCas into E.coli BL21(DE3) strain, resulting in BL21(DE3)(pCas+pTarget-N20).
Figure 2. Growth curve of BL21(DE3) (pCas+pTarget) with and without the T7 phage infection and E.coli BL21(DE3)(negative control) with the T7 phage infection in LB media.
For genome editing, we assembled a two-plasmid system, in which the cas9 gene and the sgRNA directing it to the targeted region were separated in the pCas and pTarget, respectively.
We cultivated both the BL21(DE3) (pCas+pTarget-N20) and negative control BL21(DE3), with T7 phage added in liquid LB medum when the bacteria reached logarithmic growth period. To estimate the resist efficiency of the CRISPR system, we also set a control group when BL21(DE3) (pCas+pTarget-N20) was cultivated without phage infection during the period.The results indicated the growth of BL21(DE3) (pCas+pTarget-N20) did not be affected by T7 phage, which convinced that the recombinant strain functioned quite well to resist the phage in LB, and the efficiency of resistance reached nearly 100%.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 41
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