Difference between revisions of "Part:BBa K1645998"

Latest revision as of 01:41, 24 October 2016

SgRNA targeting LacI promoter

This part is an sgRNA from the CRISPRi system and is designed to provide (d)Cas9 the specificity to target the promoter of BBa_I20260.

It can be used with Streptococcus pyogenes Cas9 and related variants. Here, we use it in conjunction with flow cytometry to demonstrate its ability to repress RFP expression.

It has been characterized through numerous experiments presented in the next section.

Characterization

Figure 1: Preliminary Exploratory Comparison of RFP Expression with (a) and without (b) a Complete sgRNA-dCas9 pair.

Figure 2: Comparison of (a) RFP with an sgRNA-dCas9 pair and (b) RFP with only dCas9 and no sgRNA

Figure 3: Comparison between (a) iptg-induced and (b) no iptg-induction CRISPRi

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K1645998 short</partinfo>
-This part contains the 20 nucleotides long sequence which is complimentary to the region within the LacI (BBa_R0010) promoter, the scaffold region which is responsible for the secondary structure of the SgRNA, and the terminator. A constitutive promoter should be placed upstream of this sgRNA in order for it to get transcribed.The suggested promoter to be used for this part is the U6 promoter. This promoter was used upstream of this part for our purposes of the project.
+This part is an sgRNA from the CRISPRi system and is designed to provide (d)Cas9 the specificity to target the promoter of BBa_I20260.
-<!-- Add more about the biology of this part here
+It can be used with Streptococcus pyogenes Cas9 and related variants. Here, we use it in conjunction with flow cytometry to demonstrate its ability to repress RFP expression.
-===Usage and Biology===
+It has been characterized through numerous experiments presented in the next section.
+==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--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--2016_Round_374(NOIPTG)vs374(IPTG).png|300px|thumb|right|Figure 3: Comparison between (a) iptg-induced and (b) no iptg-induction CRISPRi]]
+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.
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