Part:BBa_K5096056

dcas9 plasmid

The pCD017 plasmid encodes the dSpyCas9 protein, which is used in CRISPR interference (CRISPRi). dSpyCas9 is a deactivated form of Cas9 derived from Streptococcus pyogenes. dSpyCas9 lacks nuclease activity, therefore it cannot cut the DNA, but block the DNA Polymerase from initiating transcription. It does this by binding to single-guide RNA (sgRNA) to form a complex, effectively silencing the gene targeted by the sgRNA without introducing breaks in the DNA. This part was originally developed by Marshall et al. (2018). To test our CRISPRi system, we applied mathematical modeling by creating a complete set of ordinary differential equations (ODEs) to represent GFP expression dynamics. We input the initial concentrations of 1 nanomole of GFP DNA, along with reaction parameters like dCas9, sgRNA, and degradation rates. Using Simbiology’s Model Analyzer, we simulated GFP concentration over 16 hours. The graph shows a rapid initial increase in GFP expression, but around the 4-hour mark, dCas9 begins repressing the inhA gene, causing the graph to plateau as GFP expression is inhibited. We recreated the graph of RFU expression of our wetlab’s experimental GFP CRISPRi results. As the graph progresses, you would observe that at lower percentages of bound GFP DNA, the RFU values remain high, reflecting active GFP expression. However, as the binding percentage rises, particularly after the initial stages of the reaction, there will be a noticeable plateaus in RFU, correlating with the inhibition of GFP transcription. To evaluate the effectiveness of our wetlab experimentation (see CRISPRi), we utilized a line-of-best-fit between GFP expression and fluorescence to correlate the reactions, represented by the equation RFU = 6.1 x [GFP] for Lambert High School’s plate reader and RFU = 231.7 x [GFP] for the plate reader used at the Georgia Institute of Technology. By converting our CRISPRi DNA concentration to fluorescence, the similarity presented by the logarithmic shape of our model and our experimental results prove the accuracy of our wetlab. The minor discrepancies in the scales of the graphs could be attributed to potential pipetting errors or differences in the sensitivity or calibration of the plate readers we used. This graph effectively illustrates the relationship between the percentage of bound GFP DNA and both the experimental and theoretical modeled RFU values. The observed stagnation and decline in fluorescence units with the increase in bound DNA percentage demonstrates the successful application of the CRISPRi mechanism – as the graph shapes are relatively similar to the wetlab results – validating the model's predictions and emphasizing the efficiency of dCas9 in gene regulation. The differences in scales can be attributed to discrepancies in pipetting or differences with each plate reader run. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4301
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4301
  Illegal NheI site found at 1220
  Illegal NheI site found at 4060
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4301
  Illegal BamHI site found at 6339
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4301
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4301
  Illegal AgeI site found at 811
  Illegal AgeI site found at 1134
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 106