Difference between revisions of "Part:BBa K2137001"

Latest revision as of 00:14, 22 October 2019

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)

Update (Waterloo iGEM 2019): The original characterization experiments for the promoter were limited by several design flaws. In the original experiments, cells were grown in YPGal media, which is a complex medium. Because of this, it is possible that there was copper in the medium, which would have induced the promoter and prevented us from obtaining accurate characterization data for the promoter. The YPGal media was also not selective for cells containing the vector, meaning that the vector may have been lost in a portion of the cells used for characterization. Finally, the experiments characterized the promoter using only a copper ion concentration of 200 µM.

Characterization

Figure 1: Relative Fluorescence over time of Cup1-GFP fusion

Figure 2: OD-corrected Fluorescence vs. Copper Concentration

Figure 3: OD-corrected Fluorescence vs. Copper Concentration (Zoomed)

We performed a series of experiments to test the strength of the promoter using the Cup1-GFP fusion

Update (Waterloo iGEM 2019): This year, to improve the characterization of this part, we sought to obtain characterization data for the promoter after growing the cells in a defined medium, WO. This way, we could be sure there was no copper in the growth medium, and that any promoter activity was due to the added copper. This defined media also allowed for selection of cells containing the vector, ensuring that our characterization experiments did not include cells that had lost the vector. Finally, we chose to characterize the promoter by induction with a range of copper concentrations.

Methods and Materials

To characterize Cup1, strains of yeast with the plasmids were grown to an OD600 of 0.1 in YPGal media in 30 degrees Celsius on a shaker at 200 RPM and then were diluted into media and put into a plate reader. For each sample, cells were normalized 107 cells. Measurements of GFP were taken over a period of 24 hours using a Gemini XPS Microplate Reader. This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.

Update (Waterloo iGEM 2019): W303a yeast (1) were transformed with a plasmid containing the BBa_K2137001 part, a Cup1-GFP transcriptional fusion. The transformation was completed using standard protocols (2). Following transformation, cells were maintained in selective media before using the Synergy 4 BioTek plate reader to quantify expression.

Using the plate reader, we measured absorbance at 600 nm and GFP fluorescence (ex: 485 nm; em: 525 nm) in 100 µL of sample in a 96 well plate with clear bottoms and black walls. The measurements were taken 8 hours after the addition of varying amounts of copper. The absorbance and GFP fluorescence readings were corrected by subtracting the average values obtained from readings of LB-only blanks. The readings at each copper concentration were taken in triplicate from culture triplicates. The OD-corrected fluorescence was calculated by dividing the GFP fluorescence by the absorbance at 600 nm. The OD-corrected fluorescence of untransformed yeast is 1.080E3 +/- 5.905E2

Results and Discussion

The Cup1-GFP transcriptional fusion inducible promoter was another part characterized this year. This construct is the same as the Hsp104 NSC plasmid except the Gal1,10 promoter was replaced by Cup1 and CFP was replaced by GFP. This was used as a positive control to show the normal retention of our constructs with Cup1 promoters by cells. Cup1 was induced using 200 uM copper sulfate.

Figure 1. shows that over time, the yeast cells with Cup1-GFP showed relative fluorescence units 4 times induction over a control with no promoter at 6 hours and 5 times at 27 hours. Therefore, the promoter has been effectively characterized as GFP shows the expression levels produced by inducing Cup1 with 200 uM copper sulfate.

Update (Waterloo iGEM 2019): Figure 2 displays the OD-corrected fluorescence of the cells containing the Cup1-GFP fusion 8 hours after the addition of varying amounts of copper. These data represent a more robust set of experimental data characterizing the induction of the Cup1 promoter by addition of copper.

Figure 3 contains the same data as figure 2, but allows for better visualization of the data at low copper concentrations.

References

[1] Ralser, M., Kuhl, H., Ralser, M., Werber, M., Lehrach, H., Breitenbach, M., and Bernd, T. The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt. Open Biol., 2012, 2, 120093.

[2] Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res., 1992, 20, 1425.

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 292
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 941

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K2137001 short</partinfo>
@@ Line 9: / Line 8: @@
 The CUP1 promoter is inducible by adding copper to the medium (http://labs.biology.ucsd.edu/subramani/documents/121.pdf)
+<b> Update (Waterloo iGEM 2019)</b>: The original characterization experiments for the promoter were limited by several design flaws. In the original experiments, cells were grown in YPGal media, which is a complex medium. Because of this, it is possible that there was copper in the medium, which would have induced the promoter and prevented us from obtaining accurate characterization data for the promoter. The YPGal media was also not selective for cells containing the vector, meaning that the vector may have been lost in a portion of the cells used for characterization. Finally, the experiments characterized the promoter using only a copper ion concentration of 200 µM.
-<!-- Add more about the biology of this part here
-===Usage and Biology===
 ==Characterization==
-[[File:T--Waterloo--2016_Round1_374vs418.png|300px|thumb|right|Figure 1: Preliminary Exploratory Comparison of RFP Expression with (a) and without (b) a Complete sgRNA-dCas9 pair.]]
+[[File:T--Waterloo--Cup1_Characterization.png|300px|thumb|right|Figure 1: Relative Fluorescence over time of Cup1-GFP fusion]]
-[[File:T--Waterloo--2016_Round_374vs418(IPTG-BOTH.png|300px|thumb|right|Figure 2: Comparison of (a) RFP with an sgRNA-dCas9 pair and (b) RFP with only dCas9 and no sgRNA]]
+[[File:T--Waterloo--BBa K2137001-Zoom-OD-corrected-fluorescence-vs-copper.png|300px|thumb|right|Figure 2: OD-corrected Fluorescence vs. Copper Concentration]]
-[[File: T--Waterloo--2016_Round_374(NOIPTG)vs374(IPTG).png|300px|thumb|right|Figure 3: Comparison between (a) iptg-induced and (b) no iptg-induction CRISPRi]]
+[[File:T--Waterloo--BBa K2137001-OD-corrected-fluorescence-vs-copper.png|300px|thumb|right|Figure 3: OD-corrected Fluorescence vs. Copper Concentration (Zoomed)]]
-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.
+We performed a series of experiments to test the strength of the promoter using the Cup1-GFP fusion
+<b> Update (Waterloo iGEM 2019)</b>: This year, to improve the characterization of this part, we sought to obtain characterization data for the promoter after growing the cells in a defined medium, WO. This way, we could be sure there was no copper in the growth medium, and that any promoter activity was due to the added copper. This defined media also allowed for selection of cells containing the vector, ensuring that our characterization experiments did not include cells that had lost the vector. Finally, we chose to characterize the promoter by induction with a range of copper concentrations.
 ===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.
+To characterize Cup1, strains of yeast with the plasmids were grown to an OD600 of 0.1 in YPGal media in 30 degrees Celsius on a shaker at 200 RPM and then were diluted into media and put into a plate reader. For each sample, cells were normalized 107 cells. Measurements of GFP were taken over a period of 24 hours using a Gemini XPS Microplate Reader. This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.
+<b> Update (Waterloo iGEM 2019)</b>: W303a yeast (1) were transformed with a plasmid containing the BBa_K2137001 part, a Cup1-GFP transcriptional fusion. The transformation was completed using standard protocols (2). Following transformation, cells were maintained in selective media before using the Synergy 4 BioTek plate reader to quantify expression.
+Using the plate reader, we measured absorbance at 600 nm and GFP fluorescence (ex: 485 nm; em: 525 nm) in 100 µL of sample in a 96 well plate with clear bottoms and black walls. The measurements were taken 8 hours after the addition of varying amounts of copper. The absorbance and GFP fluorescence readings were corrected by subtracting the average values obtained from readings of LB-only blanks. The readings at each copper concentration were taken in triplicate from culture triplicates. The OD-corrected fluorescence was calculated by dividing the GFP fluorescence by the absorbance at 600 nm. The OD-corrected fluorescence of untransformed yeast is 1.080E3 +/- 5.905E2
 ===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.
+The Cup1-GFP transcriptional fusion inducible promoter was another part characterized this year. This construct is the same as the Hsp104 NSC plasmid except the Gal1,10 promoter was replaced by Cup1 and CFP was replaced by GFP. This was used as a positive control to show the normal retention of our constructs with Cup1 promoters by cells. Cup1 was induced using 200 uM copper sulfate.
+Figure 1. shows that over time, the yeast cells with Cup1-GFP showed relative fluorescence units 4 times induction over a control with no promoter at 6 hours and 5 times at 27 hours. Therefore, the promoter has been effectively characterized as GFP shows the expression levels produced by inducing Cup1 with 200 uM copper sulfate.
+<b> Update (Waterloo iGEM 2019)</b>: Figure 2 displays the OD-corrected fluorescence of the cells containing the Cup1-GFP fusion 8 hours after the addition of varying amounts of copper. These data represent a more robust set of experimental data characterizing the induction of the Cup1 promoter by addition of copper.
+Figure 3 contains the same data as figure 2, but allows for better visualization of the data at low copper concentrations.
+===References===
-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.
+[1] Ralser, M., Kuhl, H., Ralser, M., Werber, M., Lehrach, H., Breitenbach, M., and Bernd, T. The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt. Open Biol., 2012, 2, 120093.
-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.
+[2] Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res., 1992, 20, 1425.
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