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 GAPDH promoter(BBa_K3506020) and CnU6 promoter(BBa_K3506021).
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Constitutive double promoter system is composed of <i>GAPDH</i> promoter ([https://parts.igem.org/Part:BBa_K3506099# BBa_K3506099]
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) and <i>U6</i> promoter ([https://parts.igem.org/Part:BBa_K3506021# BBa_K3506021]).
  
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It is generally accepted that all poly(A) tail of eukaryotic cells is added by RNA polymerase II. GAPDH promoter (pGAP) is considered to be a strong constitutive promoter which is promoted by RNA polymerase II.
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It is generally accepted that <i>GAPDH</i> promoter is a strong constitutive promoter which is recognized by RNA polymerase II.
  
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RNA polymerase III promoter of the U6 small RNA gene is a common part for eukaryotic expression systems. Pol III uniquely transcribes small non-coding RNAs, including 5S rRNA , tRNAs, and other essential RNAs such as the U6 snRNA[1]. Type 3 Pol III promoters have been widely used for synthesizing small functional RNAs, such as the shRNA in RNAi applications and gRNA in CRISPR-Cas genome-editing platforms[2]. 
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<i>U6</i> promoter is used to initiate the expression of guide RNA (gRNA) in lineage tracing for eukaryotic systems. 
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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 gRNA out of the transcriptomic information by polyA tail.  
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We put GAPDH promoter in front of U6 promote(pU6).The system can not only transcribe small non-coding RNAs by U6 promoter, but also transcribe the U6 promoter and the genes downstream of U6 promoter by GAPDH promoter, which makes it possible to read the information of snRNAs out of out of transcriptomic information.  
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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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It is known that RNA polymerase III transcription product does not have polyA and cannot be captured by Oligo dT for information reading. Therefore, when you need to read Pol III transcription product information at the RNA level, you can use our dual promoter.Use GAPDH promoter to drive the transcription of RNA polymerase III promoter of the U6 small RNA gene and the genes downstream of U6 promoter.
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<i>GAPDH</i> promoter is considered to be a strong constitutive promoter. It is recognized by RNA polymerase II, which can add the polyA tail at 5' end of downstream genes.
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<i>U6</i> promoter is used to initiate the transcription of small non-coding RNAs [1] and it is recognized by RNA polymerase III, which can not add the polyA tail at 5' end of downstream genes. 
 +
 
 +
In our project, <i>U6</i> promoter is used to transcribe gRNA constitutively [2], which can be combined with Cas9. <i>GAPDH</i> promoter is used to transcribe the DNA of <i>U6</i> promoter and gRNA. So we can add the polyA tail to gRNA, which enables it to be captured by oligo dT in single cell RNA sequencing. Thus, we can obtain the lineage information together with transcriptomic information by single cell RNA sequencing.
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<partinfo>BBa_K3506095 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3506095 SequenceAndFeatures</partinfo>
  
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<b><font size"3">Properties</font></b>
 
  
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We tested the pU6 and pGAP system. The test is divided into two parts.
 
  
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First part: to test whether pGAP will affect the production and function of gRNA. We put sgRNA which target ADE2 gene downstream of U6 promoter in both experimental group and control group. Put pGAPDH upstream of U6 promoter only in experimental group. Result shows that pGAP won’t affect the production and function of gRNA, because both of the two groups turn red.(图)
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<b><font size"3">Design and Properties</font></b>
 +
 
 +
We use <i>U6</i> promoter ([https://parts.igem.org/Part:BBa_K3506021# BBa_K3506021]
 +
) to transcribe gRNA, and <i>GAPDH</i> promoter ([https://parts.igem.org/Part:BBa_K3506099# BBa_K3506099]
 +
) is used to transcribe the DNA of <i>U6</i> promoter and gRNA.
 +
 
 +
 
 +
 
 +
We tested the constitutive double promoter module. This test is divided into two steps.
 +
 
 +
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 the control group. A loss-of-function mutation in <i>ADE2</i> can result in an adenine auxotroph that forms pink colonies on YNBA paltes containing a low level of adenine, thus enabling a visual evaluation of the action of CRISPR/Cas9. Put <i>GAPDH</i> promoter upstream of <i>U6</i> promoter only in the experimental group. Results showed that both of the two groups turned pink, thus <i>GAPDH</i> promoter won’t affect the production and the function of gRNA.(Figure 1.)
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Second step: to test whether gRNA can be reverse transcribed when using oligo dT as the primer. For both the experimental group and the control group, we extracted total mRNA of these pink 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 was performed on the PCR products. There came out a correct band (208bp). Then we sequenced the products and got the anticipated results.(Figure 2.)
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[[Image:T--BNU-China--englast5.jpg|300px|thumb|center|Figure 1. A.The control group(<i>U6</i> promoter-gDNA); B.The experimental group(<i>GAPDH</i> promoter-<i>U6</i> promoter-gDNA); C and D. 4500FOA (the recipient strain).]]
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[[Image:T--BNU-China--95.jpg|300px|thumb|center|Figure 2. Gel electrophoresis results of the control group(<i>U6</i> promoter-gDNA) and the experimental group(<i>GAPDH</i> promoter-<i>U6</i> promoter-gDNA). Lane 1: Marker; Lane 2 and Lane 3: RT-PCR product of the control group; Lane 4 and Lane 5: RT-PCR product of the experimental group(208 bp).]]
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Second part: to test that whether gRNA can be reverse transcribed by oligodT. For both experimental group and control group, we extract the total mRNA of these red colonies by TRIzol. Then the mRNA was reverse transcribed by oligodT. To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specfic primers. Agarose gel electrophoresis were performed on the PCR product. There came out a correct band(图）. Then we sequenced the products and get the right result.
 
  
  
 
<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 Cryptococcus neoformans. Inserted it upstream of pU6 on PRH003 plasmid.  
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1. Construct recombinant plasmid. Get <i>GAPDH</i> promoter from the genome of <i>Cryptococcus neoformans</i>. 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 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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4. The <i>Cryptococcus 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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2.Transform the product (2.5μL) into DH5α competent cells（50μL）, coat cells on each agar plate (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones were selected for colony PCR. Expanding culture colonies at 37℃ 200rpm,extract plasmids and sequence.
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5. Pink colonies are selected and inoculated into YPD medium, then placed in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.  
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3.Use Kpn1 enzyme to linearise the plasmid and transformed it into Cryptococcus neoformans by electroporation.
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4.The C. neoformans was spreed on YNBA selection medium, and the transformants grew after being cultured in an 30℃ incubator for days. Then transferred them to a 4℃ refrigerator.  
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5.After that, red single colonies were observed. Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, and placed it in 4℃ refrigerator again.  
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6. For both the experimental group and the control group, we first extract the total mRNA of these pink colonies by TRIzol. Then the mRNA is reverse transcribed using oligo dT as the primer.
  
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6.After that, more red single colonies were observed. There was no significant difference in color between the experimental group and the control group(transformed the linearized PRH003 plasmid without GAPDH promoter). These proved that pGAP won't influence the original function of pU6 and gRNA.
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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 products to further prove the success of our design.
  
  
 
<b><font size"3">References</font></b>
 
<b><font size"3">References</font></b>
  
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【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.
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[1]Duttke S. H. (2014). RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro. The Journal of biological chemistry, 289(29), 20396–20404.
  
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【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.
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[2]Gao, Z., Herrera-Carrillo, E., & Berkhout, B. (2018). RNA polymerase II activity of type 3 pol III promoters. Molecular therapy. Nucleic acids, 12, 135–145.

Latest revision as of 03:49, 28 October 2020


Constitutive double promoter module

Constitutive double promoter system is composed of GAPDH promoter (BBa_K3506099 ) and U6 promoter (BBa_K3506021).

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

U6 promoter is used to initiate 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 gRNA out of the transcriptomic information by polyA tail.


Biology and Usage

GAPDH promoter is considered to be a strong constitutive promoter. It is recognized by RNA polymerase II, which can add the polyA tail at 5' end of downstream genes.

U6 promoter is used to initiate the transcription of small non-coding RNAs [1] and it is recognized by RNA polymerase III, which can not add the polyA tail at 5' end of downstream genes. 

In our project, U6 promoter is used to transcribe gRNA constitutively [2], which can be combined with Cas9. GAPDH promoter is used to transcribe the DNA of U6 promoter and gRNA. So we can add the polyA tail to gRNA, which enables it to be captured by oligo dT in single cell RNA sequencing. Thus, we can obtain the lineage information together with transcriptomic information by 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


Design and Properties

We use U6 promoter (BBa_K3506021 ) to transcribe gRNA, and GAPDH promoter (BBa_K3506099 ) is used to transcribe the DNA of U6 promoter and gRNA.


We tested the constitutive double promoter module. 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 the control group. A loss-of-function mutation in ADE2 can result in an adenine auxotroph that forms pink colonies on YNBA paltes containing a low level of adenine, thus enabling a visual evaluation of the action of CRISPR/Cas9. Put GAPDH promoter upstream of U6 promoter only in the experimental group. Results showed that both of the two groups turned pink, thus GAPDH promoter won’t affect the production and the function of gRNA.(Figure 1.)

Second step: to test whether gRNA can be reverse transcribed when using oligo dT as the primer. For both the experimental group and the control group, we extracted total mRNA of these pink 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 was performed on the PCR products. There came out a correct band (208bp). Then we sequenced the products and got the anticipated results.(Figure 2.)

Figure 1. A.The control group(U6 promoter-gDNA); B.The experimental group(GAPDH promoter-U6 promoter-gDNA); C and D. 4500FOA (the recipient strain).
Figure 2. Gel electrophoresis results of the control group(U6 promoter-gDNA) and the experimental group(GAPDH promoter-U6 promoter-gDNA). Lane 1: Marker; Lane 2 and Lane 3: RT-PCR product of the control group; Lane 4 and Lane 5: RT-PCR product of the experimental group(208 bp).



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 Cryptococcus 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. Pink colonies are selected and inoculated into YPD medium, then placed 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 pink colonies by TRIzol. Then the mRNA is reverse transcribed using oligo dT 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 products to further prove the success of our design.


References

[1]Duttke S. H. (2014). RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro. The Journal of biological chemistry, 289(29), 20396–20404.

[2]Gao, Z., Herrera-Carrillo, E., & Berkhout, B. (2018). RNA polymerase II activity of type 3 pol III promoters. Molecular therapy. Nucleic acids, 12, 135–145.