Part:BBa_K2137001
CUP1-GFP Transcriptional Fusion
It was necessary for us to choose promoters that expressed the constructs we were cloning into yeast cells. We wanted to characterize promoter expression with GFP before we decided which promoter to use in our system with the CFP-Hsp104 construct. Furthermore, we needed to induce a [PSI+] state by overexpression of Sup35 in the cell by inserting another copy of Sup35 into the system via a plasmid. Overall, we had two plasmids that we wanted to easily induce at any time.
We chose to characterize four main promoters that are commonly found in yeast: Gal1, Adh1, and Cup1.
The CUP1 promoter is inducible by adding copper to the medium (http://labs.biology.ucsd.edu/subramani/documents/121.pdf)
Characterization
vs374(IPTG).png)
We performed a series of experiments to demonstrate that this sgRNA when used with a dCas9 protein is able to repress RFP fluorescence when compared to controls.
Methods and Materials
To produce the data, we inoculated the appropriate E. coli strains into LB and grew it for 4hr to an OD600 of 0.4, followed by induction with IPTG at a final concentration of 1mM for 6hr. For negative controls, we did not add IPTG. Next, we diluted the culture four-fold into chilled formalin (1X PBS, 4% formaldehyde, 1.5% methanol). We used flow cytometry (Aminis ImageStream MKII) to run a sample and detected fluorescence using an excitation laser wavelength of 488nm at 200mW, as well as SSC at 1.5mW. After acquiring data from 20'000 cells in all channels, we performed analysis on the IDEAS Application v.6 software.
For Figure 1 and 2, the protocol above was modified such that the cultures after induction were incubated for 9hr instead of 6hr.
This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.
Results and Discussion
We began our experiments by first checking if our strains from last year were still viable. In Figure 1 (a) we show that the strain with an RFP and a dCas9 leads to expression of RFP, but in Figure 1 (b) we show that the strain with RFP and the complete dCas9-sgRNA pair resulted in repression of RFP fluorescence based on the spike in frequency of cells that have almost zero RFP fluorescence.
With this raw data, we were confident moving on with the part characterization and decided to extend the induction time by 3hr in an attempt to get better expression and thus more robust data. Figure 2 provides similar information to Figure 1 where we see the complete sgRNA-dCas9 pair repressing RFP fluorescence. In Figure 2 (b), we see intensities up to 2000 intensity units, but in Figure (a) we see that virtually all cells show less than 500 intensity units - an approximately four-fold repression ability.
Finally in Figure 3, we see that for (b) there is very little RFP fluorescence, but in (a) there is essentially no RFP fluorescence as well. This is indicative of IPTG having strong control over the expression of RFP in the E. coli cells.
In summary, we demonstrate this part's ability to give dCas9 the specificity to target the promoter of BBa_I20260 and effectively repress RFP fluorescence, thus characterizing this part for the first time.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 292
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 941
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