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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 41
  

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).

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.

Figure 1a: Growth situation between the BL21(DE3)(control group) (left) and the BL21(DE3) (pCas+pTarget-N20) (right) after infected by T7 phages in LB plate	Figure 1b: Growth situation between the BL21(DE3)(control group) (left) and the BL21(DE3) (pCas+pTarget-N20) (right) after infected by T7 phages in LB broth

We cultivated both the BL21(DE3) (pCas+pTarget-N20) and negative control BL21(DE3), with T7 phage added by two layer plating method and in liquid media when the bacteria reached Logarithmic growth period.

Figure 1a: Obvious plaque can be seen on both plates after phage infection. However, the plaques can hardly be observed on the plate of BL21(DE3) (pCas+pTarget-N20).

Figure 1b: The concentration of the experimental strain was significantly higher than that of the control group after introducing T7 phage, which confirmed our CRISPR/Cas9 system did work to resist this specific phage.

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.	Figure 3: Growth curve of BL21(DE3) (pCas+pTarget-N20+for+ptx) with and without the T7 phage infection and E. coli BL21(DE3)(for+ptx)(negative control) in specific MOPS medium.

Figure 2: 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%.

Figure 3: Similar experiment was conducted to examine the entire function of our Robust system in specific MOPS medium. The figure showed that the concentration of BL21(DE3) (pCas+pTarget-N20+for+ptx) did not decrease after T7 phage infection. On the other hand, lower growth rate and relatively lower final concentration of the the group with T7 phage were observed compared to the one without phage infection during this period. The encouraging growth situation of BL21(DE3) (pCas+pTarget-N20+for+ptx) in MOPS medium without phage infection indicated that the CRISPR/Cas did not affect the function of N&P Pathways. Meanwhile, CRISPR/Cas system still worked to resist T7 phage and the efficiency of resistance can reached nearly 60%. The efficiency was slightly lower than that culture in LB medium. This may due to lack of enough nutrition in the specific MOPS medium, and we will continue to optimize the concentration of formamide and phosphite of the medium to achieve a satisfactory efficiency.