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Team:SJTU-BioX-Shanghai/Project/Regulator/Integrating
From 2013.igem.org
(→Testing the Interface of CRISPRi and Light Sensors) |
(→Gathering Quantitative Data -- sgRNA targeting luciferase) |
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=IDEA!= | =IDEA!= | ||
{{Template:13SJTU_project_summary_head}} | {{Template:13SJTU_project_summary_head}} | ||
| - | Place sgRNAs under control of different light sensors. One light one sgRNA. | + | Place sgRNAs under control of different light sensors. '''One light one sgRNA'''. |
<br><br> | <br><br> | ||
Our system operates like this: | Our system operates like this: | ||
| + | <br> | ||
1. light sensors regulates sgRNAs; | 1. light sensors regulates sgRNAs; | ||
| + | <br> | ||
2. then sgRNAs regulate their corresponding genes, which is the final targets in the pathway to be optimized. | 2. then sgRNAs regulate their corresponding genes, which is the final targets in the pathway to be optimized. | ||
<br><br> | <br><br> | ||
Therefore, we are able to: | Therefore, we are able to: | ||
| - | * Regulate Genomic Genes. | + | * 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. | 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. | + | * Simultaneously controlling '''several''' genes. |
Each gene is regulated by a different light, respectively. | Each gene is regulated by a different light, respectively. | ||
| - | * Offer a easy-to-use platform. | + | * 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. | 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}} | {{Template:12SJTU_part_summary_foot}} | ||
| + | <br> | ||
=Testing CRISPRi= | =Testing CRISPRi= | ||
<br> | <br> | ||
| - | 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. | + | 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: | Then we set up a strictly controlled experiment as follows: | ||
| - | * Case: constitutive dCas9 on pRSFDuet + a constitutive gR4mRFP on pCDFDuet + constitutive mRFP on pETDuet | + | * Case: constitutive dCas9 on pRSFDuet + '''a constitutive gR4mRFP on pCDFDuet''' + constitutive mRFP on pETDuet |
| - | * Controll: constitutive dCas9 on pRSFDuet + | + | * Controll: constitutive dCas9 on pRSFDuet + ''' an empty pCDFDuet ''' + constitutive mRFP on pETDuet |
[[File:CRISPRi_test.png]] | [[File:CRISPRi_test.png]] | ||
<br> | <br> | ||
| - | As expected, mRFP repressed by CRISPRi (dCas9 and gR4mRFP) is not as red as the control. | + | As expected, '''mRFP repressed by CRISPRi (dCas9 and gR4mRFP) is not as red as the control.''' |
<br><br> | <br><br> | ||
| + | The sequence of gR4mRPF is given in our part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1026003 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. | ||
| + | <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 [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1026003 '''BBa_K1026003'''] can be an example of no-scar assembly. | ||
| + | * A reliable terminator is applied here to '''ensure the termination of sgRNA'''. | ||
| + | <br> | ||
| + | <br><br> | ||
| + | <br> | ||
| - | =Testing the Interface of CRISPRi and Light Sensors= | + | =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]] | [[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> | ||
| - | To assure that CRISPRi can be successfully combined with Light Sensors | + | 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> | <br> | ||
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[[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]] | [[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> | <br><br><br><br><br><br> | ||
| - | Since it is difficult for RFP reporter to produce qualitative results, we continue to the next phase of our project -- luciferase reporter assay. | + | 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> | ||
<br><br> | <br><br> | ||
| - | =Gathering Quantitative Data -- sgRNA targeting luciferase= | + | =Gathering '''Quantitative''' Data -- sgRNA targeting '''luciferase'''= |
<br> | <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. | + | 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> | |
<font size=4>Designing a sgRNA targeting luciferase</font size=4> | <font size=4>Designing a sgRNA targeting luciferase</font size=4> | ||
| - | Since we do not know the sequence of a working sgRNA that targets luciferase, so we designed two gR4mRFP ourselves according to those design criteria: one is closer to the start codon, whereas the other is relatively further. | + | <br> |
| - | However it is necessary for us to test these sgRNA. So we again inserted these two sgRNAs into a constitutive operon. | + | 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. We choose the proximal one for later use. | ||
| + | <br><br> | ||
| + | <font size=4>Determining the '''Working Curve''' of our Metabolic Gear Box</font size=4> | ||
| + | <br> | ||
| + | Again, it is time for our Metabolic Gear Box. | ||
<!----------------------------------------------------到这里结束---------------------------------------> | <!----------------------------------------------------到这里结束---------------------------------------> | ||
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Latest revision as of 18:10, 17 October 2013
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