Team:SJTU-BioX-Shanghai/Project/Regulator/Integrating

From 2013.igem.org

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=IDEA!=
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{{Template:13SJTU_project_summary_head}}
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Place sgRNAs under control of different light sensors. '''One light one sgRNA'''.
 +
<br><br>
 +
Our system operates like this:
 +
<br>
 +
1. light sensors regulates sgRNAs;
 +
<br>
 +
2. then sgRNAs regulate their corresponding genes, which is the final targets in the pathway to be optimized.
 +
<br><br>
 +
Therefore, we are able to:
 +
* Regulate '''Genomic''' Genes.
 +
In the past, researchers seldom accomplishes the regulation of genomic genes since it is so difficult to change endogenous promoters to be regulated by outer signals.
 +
* Simultaneously controlling '''several''' genes.
 +
Each gene is regulated by a different light, respectively.
 +
* Offer a '''easy-to-use''' platform.
 +
Users of our Metabolic Gear Box can optimize their own desired pathway after making some minor changes to a vector of sgRNAs.
 +
{{Template:12SJTU_part_summary_foot}}
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<br>
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=From CRISPR to CRISPRi=
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=Testing CRISPRi=
<br>
<br>
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<font size=4>CRISPR</font size=4>
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To test CRISPRi, we have constructed a '''constitutive''' dCas9 on pRSFDuet, a '''constitutive sgRNA targeting mRFP (gR4mRFP)''' on pCDFDuet and a constitutive mRFP on pETDuet.
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[[File:CRISPR_genome_editing.png|thumb|300px|right|CRISPR Genome Editing (Jinek et al., 2012)]]
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Then we set up a strictly controlled experiment as follows:
 +
* Case:    constitutive dCas9 on pRSFDuet + '''a constitutive gR4mRFP on pCDFDuet''' + constitutive mRFP on pETDuet
 +
* Controll: constitutive dCas9 on pRSFDuet + '''      an empty pCDFDuet          ''' + constitutive mRFP on pETDuet
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[[File:CRISPRi_test.png]]
<br>
<br>
-
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and <b>CRISPR-associated system (Cas)</b> widely exist in Bacteria and Archea, endowing the cell with <b>adaptive immunity</b> to foreign DNAs. The immune process could be divided into three stages:<br>
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As expected, '''mRFP repressed by CRISPRi (dCas9 and gR4mRFP) is not as red as the control.'''
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* <b>Adaptive Stage</b>.    Fragments of foreign DNA (named protospacers) is incorporated into the proximal end of CRISPR.<br>
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<br><br>
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* <b>Expression Stage</b>.   Precursor CRISPR RNAs (pre-crRNAs) are transcribed from CRISPR. Pre-crRNAs are then cleaved to produce <b>crRNAs</b>.<br>
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The sequence of gR4mRPF is given in our part BBa_K1026003 . This sequence comes from the following literature: QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
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* <b>Interference Stage</b>.    crRNA complements with target DNA (protospacers), forming a complex that is recognized and bound by Cas nuclease (Csn). Csn excises the target, leaving the target vulnerable to successive degradation.
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<br><br>
 +
And <font size=4>'''CAUTION'''</font> that:
 +
* There shall '''not''' be extra nucleotides between promoter and sgRNA itself.
 +
gRNA shall be exactly transcribed out. Any extra nucleotides would potentially jeopardize the normal function of the gRNA. Therefore, '''BioBrick scars are not allowed here'''. Instead of cut-and-ligation, inverse PCR or In-Fusion is preferred for the assembly task.
 +
Our part '''BBa_K1026003''' can be an example of no-scar assembly.
 +
* A reliable terminator is applied here to '''ensure the termination of sgRNA'''.
<br>
<br>
-
Three types of CRISPR system have already been revealed, of which Type II CRISPR system is the <b>simplest</b> – one sole protein, <b>Cas9</b> (formerly named Csn1) is capable of all tasks once crRNA is complemented by <b>trans-activating crRNA (tracrRNA)</b>. Due to this simplicity, Type II CRISPR/Cas can be ectopically expressed as a tool for sequence-specific genome editing (Jinek et al., 2012). And to further simplify the tool, Jinek et al. linked crRNA and tracr RNA together, successfully creating <b>small guide RNA (sgRNA)</b> as an equivalent (Jinek et al., 2012).
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<br><br>
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<br><br><br>
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<font size=4>CRISPRi</font size=4><br>
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[[File:CRISPRi.png|250px|right|thumb|CRISPRi Mechanism]]
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<br>
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With its <b>endonuclease activity crippled</b>, Cas9 can be used to hinder RNA polymerase and transcript elongation. Together with a proper sgRNA, Cas9 possessing such defect (named dCas9) can be used as an effective tool in expression interrogation – <b>CRISPR interference</b>, abbreviated as CRISPRi (Qi et al., 2013).
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<br>
<br>
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CRISPRi is promising tool for expression regulation, <b>versatile and easy-to-use</b>. Generally, a small shift in sgRNA would be enough for a new target.
 
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<br><br><br>
 
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=Key Parts of CRISPRi=
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=Testing the '''Interface''' of CRISPRi and Light Sensors=
 +
[[File:SgRNA_operons.png|thumb|300px|right|sgRNAs regulated by light sensors.    Up: Red light controlled sgRNA; Medium: Green light controlled sgRNA; Down: Blue light controlled sgRNA]]
<br>
<br>
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There are only 2 parts to be expressed -- dCas9 and sgRNA. Only <font size=4>2</font size=4>!
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To assure that CRISPRi can be successfully combined with Light Sensors, we constructed '''sgRNAs targeting mRFP that are regulated by <font color=red>red</font>, <font color=green>green</font> and <font color=blue>blue light sensors</font>, respectively''':
 +
* POmpC is a promoter negatively regulated by Red Light.
 +
* K1026009, one of our parts, is a artificial promoter negatively regulated by green light.
 +
* FixK2 is a promoter negatively regulated by Blue Light.
 +
<br>
 +
 
 +
And next, it is show time for our Metabolic Gear Box!
 +
[[File:Metabolic_Gear_Box_with_Culture.JPG|thumb||700px|A Metabolic Gear Box in work! With bacteria culture! And in a thermostat incubator!]]
 +
[[File:Metabolic_Gear_Box_Controlling_System.JPG|thumb|700px|A Metabolic Gear Box Receiving Feed-forward Commands.    Researchers will now send commands of desired expression level to the computer. Then these commands will be transferred to the Box]]
 +
<br><br><br><br><br><br>
 +
Since it is difficult for RFP reporter to accurately produce qualitative results, we continue to the next phase of our project -- luciferase reporter assay.
<br><br>
<br><br>
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<font size=4>dCas9</font size=4>
 
<br><br>
<br><br>
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As the signature protein of the type II CRISPR/Cas, Cas9, does not show any detectable similarity to any proteins in Type I and Type III systems, in that it is sufficient both to generate crRNA and to cleave the target DNA. This large protein (about 1000 amino acids) contains two predicted nuclease domains -- the N-terminal '''RuvC-like nuclease''' (RNAse H fold) and the '''HNH (McrA-like) nuclease''' domain that is located in the middle of the protein (Makarova et al., 2011), each of which would cleave one DNA strand.
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To acquire dCas9, one point mutation is conducted on each nuclease, respectively, namely, '''D10A and H841A''' (Qi et al., 2013).
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=Gathering '''Quantitative''' Data -- sgRNA targeting '''luciferase'''=
 +
<br>
 +
In order to know '''whether our system provides a wide enough range of continuous adjustment''', we constructed sgRNAs targeting luciferase (gR4luciferase) gene. And again, these gR4luciferase are under controll of red, green and blue light, respectively.
<br><br>
<br><br>
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<font size=4>sgRNA</font size=4>
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<font size=4>Designing a sgRNA targeting luciferase</font size=4>
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[[File:SgRNA.png|thumb|250px|right|Structure of sgRNA]]
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<br>
<br>
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If dCas9 acts as an executive, than sgRNA is the director of CRISPRi.
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Since we do not know the sequence of a working sgRNA that targets luciferase, so we designed two gR4mRFP that complements the coding sequence of luciferase gene ourselves according to those design criteria: one is '''closer to the start codon (the proximal one)''', whereas the other is relatively further (the '''distal one''').
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An sgRNA constitutes three parts:
+
However it is necessary for us to test these sgRNA. So we again inserted these two sgRNAs into a constitutive operon. After conducting a luciferase reporter assay with a Beyotime kit, it comes out that the repression effect of these two sgRNAs are 80% and 60%, respectively.
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* '''Base-Pairing Region'''
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Therefore, we choose the proximal one for later use.
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A 20 nt complementary region for specific DNA binding.
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* dCas9 '''Handle'''
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a 42 nt hairpin for Cas9 binding
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* A '''terminator''' derived from S. pyogenes, 42nt
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<br>
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<b><font size=2.5>Design Criteria</font></b> (Qi et al., 2013)
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* Binding specificity is determined by both sgRNA-DNA base pairing and a <b>protospacer adjacent motif (PAM), NGG</b>, upstream of target.
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* Generally, the optimal length of the complementary region is <b>20nt</b>.
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* If a sgRNA targets coding sequence, it has to be <b>complementary to the non-template (NT) strand</b>. But if a sgRNA targets the promoter, either strand would be acceptable.
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* If a sgRNA targets coding sequence, generally CRISPRi would work better if the complementary region is <b>closer to the promoter</b>.
+
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* The first 7 nt might be a "seed region" for binding, since any single mutation of  dramatically decreased repression.
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<br><br>
<br><br>
-
 
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<font size=4>Determining the '''Working Curve''' of our Metabolic Gear Box</font size=4>
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<html>
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<h1 style="color:grey;">References</h1>
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<p style="color:grey;">
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<br>
<br>
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LEVSKAYA, A., CHEVALIER, A. A., TABOR, J. J., SIMPSON, Z. B., LAVERY, L. A., LEVY, M., DAVIDSON, E. A., SCOURAS, A., ELLINGTON, A. D., MARCOTTE, E. M. & VOIGT, C. A. 2005. Synthetic biology: engineering Escherichia coli to see light. Nature, 438, 441-2.
+
Again, it is time for our Metabolic Gear Box.
-
<br/><br/>
+
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TABOR, J. J., LEVSKAYA, A. & VOIGT, C. A. 2011. Multichromatic control of gene expression in escherichia coli. Journal of Molecular Biology, 405, 315-324.
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-
</p></html>
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<!----------------------------------------------------到这里结束--------------------------------------->
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Revision as of 00:29, 28 September 2013

IDEA!

Place sgRNAs under control of different light sensors. One light one sgRNA.

Our system operates like this:
1. light sensors regulates sgRNAs;
2. then sgRNAs regulate their corresponding genes, which is the final targets in the pathway to be optimized.

Therefore, we are able to:

  • Regulate Genomic Genes.

In the past, researchers seldom accomplishes the regulation of genomic genes since it is so difficult to change endogenous promoters to be regulated by outer signals.

  • Simultaneously controlling several genes.

Each gene is regulated by a different light, respectively.

  • Offer a easy-to-use platform.

Users of our Metabolic Gear Box can optimize their own desired pathway after making some minor changes to a vector of sgRNAs.


Testing CRISPRi


To test CRISPRi, we have constructed a constitutive dCas9 on pRSFDuet, a constitutive sgRNA targeting mRFP (gR4mRFP) on pCDFDuet and a constitutive mRFP on pETDuet. Then we set up a strictly controlled experiment as follows:

  • Case: constitutive dCas9 on pRSFDuet + a constitutive gR4mRFP on pCDFDuet + constitutive mRFP on pETDuet
  • Controll: constitutive dCas9 on pRSFDuet + an empty pCDFDuet + constitutive mRFP on pETDuet

CRISPRi test.png
As expected, mRFP repressed by CRISPRi (dCas9 and gR4mRFP) is not as red as the control.

The sequence of gR4mRPF is given in our part BBa_K1026003 . This sequence comes from the following literature: QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.

And CAUTION that:

  • There shall not be extra nucleotides between promoter and sgRNA itself.

gRNA shall be exactly transcribed out. Any extra nucleotides would potentially jeopardize the normal function of the gRNA. Therefore, BioBrick scars are not allowed here. Instead of cut-and-ligation, inverse PCR or In-Fusion is preferred for the assembly task. Our part BBa_K1026003 can be an example of no-scar assembly.

  • A reliable terminator is applied here to ensure the termination of sgRNA.





Testing the Interface of CRISPRi and Light Sensors

sgRNAs regulated by light sensors. Up: Red light controlled sgRNA; Medium: Green light controlled sgRNA; Down: Blue light controlled sgRNA


To assure that CRISPRi can be successfully combined with Light Sensors, we constructed sgRNAs targeting mRFP that are regulated by red, green and blue light sensors, respectively:

  • POmpC is a promoter negatively regulated by Red Light.
  • K1026009, one of our parts, is a artificial promoter negatively regulated by green light.
  • FixK2 is a promoter negatively regulated by Blue Light.


And next, it is show time for our Metabolic Gear Box!

A Metabolic Gear Box in work! With bacteria culture! And in a thermostat incubator!
A Metabolic Gear Box Receiving Feed-forward Commands. Researchers will now send commands of desired expression level to the computer. Then these commands will be transferred to the Box







Since it is difficult for RFP reporter to accurately produce qualitative results, we continue to the next phase of our project -- luciferase reporter assay.



Gathering Quantitative Data -- sgRNA targeting luciferase


In order to know whether our system provides a wide enough range of continuous adjustment, we constructed sgRNAs targeting luciferase (gR4luciferase) gene. And again, these gR4luciferase are under controll of red, green and blue light, respectively.

Designing a sgRNA targeting luciferase
Since we do not know the sequence of a working sgRNA that targets luciferase, so we designed two gR4mRFP that complements the coding sequence of luciferase gene ourselves according to those design criteria: one is closer to the start codon (the proximal one), whereas the other is relatively further (the distal one). However it is necessary for us to test these sgRNA. So we again inserted these two sgRNAs into a constitutive operon. After conducting a luciferase reporter assay with a Beyotime kit, it comes out that the repression effect of these two sgRNAs are 80% and 60%, respectively. Therefore, we choose the proximal one for later use.

Determining the Working Curve of our Metabolic Gear Box
Again, it is time for our Metabolic Gear Box.


Template:12SJTU footer

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