Difference between revisions of "Part:BBa K1645998"
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<partinfo>BBa_K1645998 short</partinfo>
 
<partinfo>BBa_K1645998 short</partinfo>
  
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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.
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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.
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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.
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It has been characterized through numerous experiments presented in the next section.
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==Characterization==
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[[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.]]
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[[File: T--Waterloo--2016_Round_374vs418(IPTG-BOTH.png|300px|thumb|right|Figure 1: A Comparison of GFP and RFP expression from the Dual Colour Plasmid]]
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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.  
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===Methods and Materials===
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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. 
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This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.
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===Results and Discussion===
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Figure 1. shows that both RFP and GFP are fluorescing, though at different intensities. Overall, GFP's intensity data averages at 502 intensity units and RFP's intensity data averages at 139 intensity units. This means that GFP fluoresces approximately 3.5x more intensely than RFP. Figure 2 shows the frequency at which cells fluoresce at a particular intensity for GFP on the right and RFP on the left.
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In all, further experiments can provide more precise measurements of GFP and RFP fluorescence, but we present here adequate fluorescence data for other teams to understand the behavior of the Dual Colour Plasmid in
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1645998 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1645999 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1645998 parameters</partinfo>
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<partinfo>BBa_K1645999 parameters</partinfo>
 
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Revision as of 03:14, 20 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 1: A Comparison of GFP and RFP expression from the Dual Colour Plasmid

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.

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

Figure 1. shows that both RFP and GFP are fluorescing, though at different intensities. Overall, GFP's intensity data averages at 502 intensity units and RFP's intensity data averages at 139 intensity units. This means that GFP fluoresces approximately 3.5x more intensely than RFP. Figure 2 shows the frequency at which cells fluoresce at a particular intensity for GFP on the right and RFP on the left.

In all, further experiments can provide more precise measurements of GFP and RFP fluorescence, but we present here adequate fluorescence data for other teams to understand the behavior of the Dual Colour Plasmid in


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1708
    Illegal AgeI site found at 1820
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 706