Difference between revisions of "Part:BBa K3506095"
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<partinfo>BBa_K3506095 short</partinfo>
 
<partinfo>BBa_K3506095 short</partinfo>
  
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Constitutive double promoter system is composed of <i>GAPDH</i> promoter ([https://parts.igem.org/Part:BBa_K3506020 BBa_K3506020]) and CnU6 promoter([https://parts.igem.org/Part:BBa_K3506021 BBa_K3506021]) .
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Constitutive double promoter system is composed of <i>GAPDH</i> promoter (BBa_K3506020) and U6 promoter (BBa_K3506021).
  
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It is generally accepted that <i>GAPDH</i> promoter (pGAP) is considered to be a strong constitutive promoter which is used by RNA polymerase II.
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It is generally accepted that <i>GAPDH</i> promoter is considered to be a strong constitutive promoter which is recognized by RNA polymerase II.
  
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U6 promoter(pU6) is used to drive the expression of homing guide RNA（hgRNA） in lineage tracing for eukaryotic systems. 
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<i>U6</i> promoter is used to drive the expression of guide RNA（gRNA） in lineage tracing for eukaryotic systems. 
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We put pGAP in the upstream of pU6. The system can read the information of sgRNAs out of the transcriptomic information by polyA tail.  
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We put <i>GAPDH</i> promoter in the upstream of <i>U6</i> promoter. The system can read the information of gRNAs out of the transcriptomic information by polyA tail.  
  
  
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<b><font size"3">Biology and Usage</font></b>
 
<b><font size"3">Biology and Usage</font></b>
  
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pGAP is considered to be a strong constitutive promoter which is promoted by RNA polymerase II.
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<i>GAPDH</i> promoter is considered to be a strong constitutive promoter which is recognized by RNA polymerase II.
  
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RNA polymerase III uniquely transcribes small non-coding RNAs, including 5S rRNA , tRNAs, and other essential RNAs such as the U6 snRNA[1]. pU6 is used to drive the expression of sgRNA in lineage tracing for eukaryotic systems. 
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<i>U6</i> promoter promoter is used to drive the transcription of small non-coding RNAs [1] and it is recognized by RNA polymerase III. 
  
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In our project, we use pU6 to transcribe small non-coding RNAs, hgRNA in CRISPR-Cas genome-editing system[2]. We use pGAP to transcribe the hgRNA and add a polyA tail. Therefore the hgRNA can not only work with CRIAPR/Cas9 system, but also work as barcodes. It enables us to read the lineage information out of transcriptomic information.
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In our project, we use <i>U6</i> promoter to transcribe functional hgRNA without polyA tail[2]. We use <i>GAPDH</i> promoter to transcribe hgRNA and add a polyA tail. Therefore, hgRNA can not only work with CRISPR/Cas9 system, but also be reverse transcribed by oligodT. It enables us to read the lineage information in hgRNA together with the transcriptomic information when single cell RNA sequencing.
  
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It is known that RNA polymerase III transcription product does not have polyA and cannot be captured by Oligo dT when reading transcriptomic information. Therefore, when you need to confirm the information of Pol III transcription products together with transcriptomic information, you can use this double promoter system. Use pGAP to drive the transcription of RNA polymerase III genes and add polyA tail as well.
 
  
  
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<b><font size"3">Properties</font></b>
 
<b><font size"3">Properties</font></b>
  
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We tested the pU6 and pGAP systems. The test is divided into two steps.
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We tested the constitutive double promoter system. This test is divided into two steps.
  
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First step: to test whether pGAP will affect the production and the function of gRNA. We put gRNA targeting <i>ADE2</i> gene downstream of pU6 in both the experimental group and control group. Put pGAP upstream of U6 promoter only in the experimental group. Results showed that both of the two groups turn red, thus pGAP won’t affect the production and the function of gRNA.
+
First step: to test whether <i>GAPDH</i> promoter will affect the production and the function of gRNA. We put gRNA targeting <i>ADE2</i> gene downstream of <i>U6</i> promoter in both the experimental group and control group. Put <i>GAPDH</i> promoter upstream of <i>U6</i> promoter only in the experimental group. Results showed that both of the two groups turn red, thus <i>GAPDH</i> promoter won’t affect the production and the function of gRNA.
  
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Second step: to test that whether gRNA can be reverse transcribed using oligo dT as the primer. For both the experimental group and the control group, we extracted total mRNA of these purified red colonies by TRIzol. Then the mRNA was reverse transcribed using oligo dT as the primer. To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specific primers. Agarose gel electrophoresis were performed on the PCR product. There came out a correct band. Then we sequenced the products and get the anticipated results.
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Second step: to test that whether gRNA can be reverse transcribed using oligo dT as the primer. For both the experimental group and the control group, we extracted total mRNA of these purified red colonies by TRIzol. Then the mRNA was reverse transcribed using oligo dT as the primer. To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specific primers. Agarose gel electrophoresis were performed on the PCR product. There came out a correct band (208bp). Then we sequenced the products and get the anticipated results.
  
  
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<b><font size"3">Experimental approach</font></b>
 
<b><font size"3">Experimental approach</font></b>
  
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1.Construct recombinant plasmid. Get pGAP from the genome of <i>Cryptococcus neoformans</i>. Insert it upstream of pU6 on PRH003 plasmid.  
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1.Construct recombinant plasmid. Get <i>GAPDH</i> promoter from the genome of Cryptococcus neoformans. Insert it upstream of <i>U6</i> promoter on PRH003 plasmid.  
  
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2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on each agar plate (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.  
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2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.  
  
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3.Use Kpn1 enzyme to linearise the plasmids and transform them into <i>Cryptococcus neoformans</i> by electroporation.   
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3.Use Kpn1 enzyme to linearise the plasmids and transform them into Cryptococcus neoformans by electroporation.   
  
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4.The <i>C. neoformans</i> is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.  
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4.The C. neoformans is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.  
  
 
5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.  
 
5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.  
  
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6.For both the experimental group and the control group, we first extract the total mRNA of these red colonies by TRIzol. Then the mRNA is reverse transcribed using oligo dT as the primer.
+
6.For both the experimental group and the control group, we first extract the total mRNA of these red colonies by TRIzol. Then the mRNA is reverse transcribed using oligodT as the primer.
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+
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7.To test whether gRNA can be transcribed, we perform PCR on the reverse transcription products by two specfic primers. Then sequencing the PCR product to further prove the success of our design.
+
  
 +
7.To test whether gRNA can be reverse transcribed, we perform PCR on the reverse transcription products by two specfic primers. Then sequencing the PCR product to further prove the success of our design.
  
  

Revision as of 08:22, 27 October 2020


Constitutive double promoter module

Constitutive double promoter system is composed of GAPDH promoter (BBa_K3506020) and U6 promoter (BBa_K3506021).

It is generally accepted that GAPDH promoter is considered to be a strong constitutive promoter which is recognized by RNA polymerase II.

U6 promoter is used to drive the expression of guide RNA（gRNA） in lineage tracing for eukaryotic systems.  We put GAPDH promoter in the upstream of U6 promoter. The system can read the information of gRNAs out of the transcriptomic information by polyA tail.


Biology and Usage

GAPDH promoter is considered to be a strong constitutive promoter which is recognized by RNA polymerase II.

U6 promoter promoter is used to drive the transcription of small non-coding RNAs [1] and it is recognized by RNA polymerase III. 

In our project, we use U6 promoter to transcribe functional hgRNA without polyA tail[2]. We use GAPDH promoter to transcribe hgRNA and add a polyA tail. Therefore, hgRNA can not only work with CRISPR/Cas9 system, but also be reverse transcribed by oligodT. It enables us to read the lineage information in hgRNA together with the transcriptomic information when single cell RNA sequencing.


Sequence and Feature


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 436


Properties

We tested the constitutive double promoter system. This test is divided into two steps.

First step: to test whether GAPDH promoter will affect the production and the function of gRNA. We put gRNA targeting ADE2 gene downstream of U6 promoter in both the experimental group and control group. Put GAPDH promoter upstream of U6 promoter only in the experimental group. Results showed that both of the two groups turn red, thus GAPDH promoter won’t affect the production and the function of gRNA.

Second step: to test that whether gRNA can be reverse transcribed using oligo dT as the primer. For both the experimental group and the control group, we extracted total mRNA of these purified red colonies by TRIzol. Then the mRNA was reverse transcribed using oligo dT as the primer. To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specific primers. Agarose gel electrophoresis were performed on the PCR product. There came out a correct band (208bp). Then we sequenced the products and get the anticipated results.


Experimental approach

1.Construct recombinant plasmid. Get GAPDH promoter from the genome of Cryptococcus neoformans. Insert it upstream of U6 promoter on PRH003 plasmid.

2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.

3.Use Kpn1 enzyme to linearise the plasmids and transform them into Cryptococcus neoformans by electroporation.

4.The C. neoformans is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.

5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.

6.For both the experimental group and the control group, we first extract the total mRNA of these red colonies by TRIzol. Then the mRNA is reverse transcribed using oligodT as the primer.

7.To test whether gRNA can be reverse transcribed, we perform PCR on the reverse transcription products by two specfic primers. Then sequencing the PCR product to further prove the success of our design.


References

[1]Duttke, S. H C . RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro.[J]. Journal of Biological Chemistry, 2014, 289(29):20396.

[2]Gao Z, Herrera-Carrillo E, Berkhout B. RNA Polymerase II Activity of Type 3 Pol III Promoters. Mol Ther Nucleic Acids. 2018 Sep 7;12:135-145.