Primary or Secondary Structure Design?

Construct Alpha had a variable loop region that was built by selecting regions of the primary sequence of the target RNA and complementing these regions with our variable regions (in text format). We then tested these sequences for hairpin loop-like secondary structure using NUPACK.

Construct Gamma had a variable loop region that was built by selecting regions of the secondary structure of the target RNA and complementing these regions with our variable regions (from NUPACK-generated structures). We then tested these sequences for hairpin loop-like secondary structure using NUPACK.

We measured the fluorescence/OD600 of the two constructs, Alpha (BBa_K2841503) and Gamma (BBa_K2841501), in a 96-well plate reader (TECAN F500) to observe which method gives the best output (Fig. 2). Results were blanked against M9 minimal media and M9 minimal media with ATC (to be subtracted from data for results where we induced with ATC only). Results of fluorescence/OD600 at a time point of 18 hours (maximum OD600) shows:
• Construct Alpha shows 2.1-2.5-fold upregulation of GFP when induced
• Construct Gamma shows 1.8-fold upregulation of GFP when induced
• The Gamma construct cells did not achieve an OD600 as high as that of other cells upon induction with ATC.

Figure 2: Induction by ATC shows differences in fluorescence for constructs Alpha (AI + AN) and Gamma (LI + LN). Error bars show standard deviation. Number of replicas = 3. The positive control were cells constitutively expressing GFP (due to a deletion of lacI in the chromosome), the negative control were cells containing pSB1AK8 with appropriate antibiotics. (A) Fluorescence/OD600 over time. (B) OD600 over time. (C) Fluorescence over time.

A Shapiro-Wilk test for normality showed the data were not normally distributed (p = 0.000) (Fig. 3A). A Mann-Whitney U test was conducted to test for statistical significance. The data was statistically significant for both constructs, Alpha and Gamma (p = 0.000) (Fig. 3B).

Figure 3: Statistical analysis of the raw fluorometry data for constructs Alpha and Gamma. (A) Shapiro-Wilk test for normality showed data were not normally distributed. Therefore, we needed to run a non-parametric test. (B) A Mann-Whitney U test was conducted to test for the statistical significance of our data. The data shows both constructs expressed statistically significant data (p = 0.000). (C) A Mann-Whitney U test for categories across constructs shows statistical significance, indicating that the exact sequence of our variable sRNA-binding regions gives significantly different data based on the primary sequence of these regions.

Taken together these data show that the synthetic guide RNA constructs, Alpha and Gamma, are capable of significant induction of gene regulation via their ability to guide dCas9 to its target DNA to alter gene expression.

Our data also show statistically significant differences in fluorescence/OD600 based on the construct that is used; even if targeting different regions of the same RNA (in our case, legiolysin) (Fig. 3C) (p = 0.000). This indicates that there is a significant difference in fluorescence highly dependent on the primary sequence of the variable regions of the guide RNA. It is also worth noting that the data across constructs for non-induction are also statistically significant. Thus, there is significant differences in background fluorescence and in induced fluorescence based on the primary sequence of the guide RNA.

Team:Warwick/Results

Results

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