Part:BBa_K3544888

pGAP-NLS-yeGFP-CRISPR(Csy4)-DsRed-tCYC1

This is a composite parts of yeGFP and DsRed using CRISPR as a spacer sequence.

BBa_K3544105 was constructed on the pY26TEF-GDP, and the cleavage efficiency of CRISPR-Csy4 system (BBa_K3544888 & BBa_K3544503) in S. cerevisiae was measured to be 98.17%.

The transcriptional level was measured by qPCR in a CFX Connect Real-Time PCR Detection System using low-profile 0.2 ml 8-tube strips. Three pairs of gene-specific primers locating in different gene sites were picked from NCBI for this experiment to reduce errors in detecting from non-specific amplification (Fig 11). F1 and R1 match with NLS-yeGFP, F3 and R3 match with DsRed. Meanwhile, F2 matches with the downstream of NLS-yeGFP and R2 matches with the upstream sequence of DsRed. And we also choose TDH as a reference gene for normalization.

Fig 1. Primers for RT-qPCR

Firstly, the original data of the gene transcription level was determined in a CFX Connect Real-Time PCR Detection System using 8-tube strips (Fig 12). The primary results showed in Cq value indicated there were confirmed differences in transcriptional level of yeGFP-CRISPR-DsRed.

Fig 2. Original data of qPCR

It was clear that using primers F2 and R2, the Cq value was apparently less than using primers F1, R1 and primers F3, R3. This suggests high cleavage efficiency of Csy4 had happened in CRISPR.

In order to display results in a visible and understandable form, we further processed original data. It’s easy to know that many factors may affect gene transcription level of S. cerevisiae, for example, culture time and density of yeast. So, we normalized gene (yeGFP, DsRed and yeGFP-28nt-dsRED) transcriptional level. To be more precisely, Cq values of three gene fragments (yeGFP, DsRed and yeGFP-CRISPR-dsRED) minus the mean Cq Value of TDH gene (mean value is 22.01) separately and calculated each mean values of difference of three gene fragments (Fig 13).

Fig 3. Normalized value of three gene fragments in qPCR

To validate the cleavage efficiency of Csy4, we regarded the transcription level of yeGFP as a background of those three gene fragments considering the transcriptional characteristic of polycistron. Then, we use the Δvalue of those three fragments minus the mean Δvalue of yeGFP gene (mean value = 3.57) separately and calculated each mean values of difference of those three fragments (Fig 14).

Fig 4. Δ(Δvalue) of three gene fragments in qPCR

Based on principle of qPCR which is exponential amplification, we regarded 2 as a base number and the mean values of three fragments as a power to get relatively normalized transcriptional level (Fig 15).

Fig 5. 2-Δ (Δvalue) of three gene fragments in qPCR

Finally, those relatively normalized values were represented as a histogram which is more visible and understandable (Fig 16).

Fig 6. Histogram of qPCR

Based from the relatively normalized results of those three gene fragments, it’s easy to know that the cleavage efficiency of Csy4 is about 98.17%.

For more imformation, please view https://2020.igem.org/Team:SCU-China/Design.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2714
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2346
  Illegal NgoMIV site found at 2867
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1156
  Illegal SapI site found at 238