Difference between revisions of "Part:BBa K3506022"
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<partinfo>BBa_K3506022 short</partinfo>
 
<partinfo>BBa_K3506022 short</partinfo>
  
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GAL7 promoter can be induced by galactose in Cryptococcus neoformans. It is the first inducible promoter characterized in C. neoformans. 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&#12304;1&#12305;. But it is known that RNA polymerase III transcription product does not have polyA and cannot be captured by Oligo dT for information reading.We designed this composite part to read the sequence information downstream of the U6 promoter in real time and at RNA level through the drive of GAL7 promoter.
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Inducible dual promoter system is composed of GAL7(BBa_K3506424) and CnU6(BBa_K3506021).
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GAL7 promoter can be induced by galactose in Cryptococcus neoformans. It is the first inducible promoter characterized in C. neoformans.  
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U6 promoter is used to drive the expression of homing guide RNA（hgRNA） in lineage tracing for eukaryotic systems. 
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We put GAPDH promoter in the upstream of U6 promote.The system can read the information of snRNAs out of out of transcriptomic information by polyA tail.  
  
 
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===Usage and Biology===
 
===Usage and Biology===
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It is known that the RNA polymerase III transcription product does not have polyA, nor can it be captured by Oligo dT for information reading. Therefore, when you need to read Pol III transcription product information at RNA level, you can use our dual promoter. Of course, the advantage of this compound part is that it can induce the expression of GAL7 by lactose, obtain the transcription information of the downstream sequence, and realize the function of reading information in real time.
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GAL7 promoter can be induced by galactose in Cryptococcus neoformans and it is promoted 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]. U6 promoter is used to drive the expression of sgRNA in lineage tracing for eukaryotic systems. 
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In our project, we use U6 promoter to transcribe small non-coding RNAs, hgRNA in CRISPR-Cas genome-editing system[2]. We use GAL7 promoter to transcribe the hgRNA and add a polyA tail at specific time. So the hgRNA can not only work with CRIAPR/Cas system, but also work as barcode. It enable us to read the lineage information out of transcriptomic information.
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It is known that the RNA polymerase III transcription product does not have polyA, nor can it be captured by Oligo dT for information reading. Therefore, you can use our double promoter system when you need to read the expression levels or information of Pol III transcription product together with transcriptomic information at specific time. This is very significant for knowing the functions and influences of this kind of RNA. Of course, this compound part can be induced, it indicates the possibility to read snRNA information at specific time or tissue.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3506022 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3506022 SequenceAndFeatures</partinfo>
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<b><font size="3">Properties</font></b>
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we tested the pU6 and pGAL7 system. The test is divided into two parts.
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First part: to test whether pGAL7 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 pGAL7 upstream of U6 promoter only in experimental group. Result shows that pGAL7 won’t affect the production and function of gRNA, because both of the two groups turn red.(Figure1)
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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(Figure2). Then we sequenced the products to prove the success of our engineer further.
  
 
<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
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3.Use Kpn1 enzyme to linearise the plasmid and transformed it into Cryptococcus neoformans by electroporation.   
 
3.Use Kpn1 enzyme to linearise the plasmid and transformed it into Cryptococcus neoformans by electroporation.   
 
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.  
 
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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5.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.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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6.For both experimental group and control group, we selected red colonies, induced by galactose for 30mins, extract the total mRNA by TRIzol. Then the mRNA was reverse transcribed by oligodT.
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7.To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specfic primers. Then sequence the PCR product to proved the success of our design further.
  
 
<b><font size="3">References</font></b>
 
<b><font size="3">References</font></b>

Revision as of 11:24, 22 October 2020


Inducible double promoter system

Inducible dual promoter system is composed of GAL7(BBa_K3506424) and CnU6(BBa_K3506021). GAL7 promoter can be induced by galactose in Cryptococcus neoformans. It is the first inducible promoter characterized in C. neoformans. U6 promoter is used to drive the expression of homing guide RNA（hgRNA） in lineage tracing for eukaryotic systems.  We put GAPDH promoter in the upstream of U6 promote.The system can read the information of snRNAs out of out of transcriptomic information by polyA tail.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 402
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 742


Properties

we tested the pU6 and pGAL7 system. The test is divided into two parts. First part: to test whether pGAL7 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 pGAL7 upstream of U6 promoter only in experimental group. Result shows that pGAL7 won’t affect the production and function of gRNA, because both of the two groups turn red.(Figure1) 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(Figure2). Then we sequenced the products to prove the success of our engineer further.

Experimental approach

1.Construct recombinant plasmid. Get pGAL7 from the PYES2 plasmid. Inserted it upstream of pU6 on PRH003 plasmid. Ligate the fragments by in-fusion cloning. 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. 3.Use Kpn1 enzyme to linearise the plasmid and transformed it into Cryptococcus neoformans by electroporation. 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. 5.Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, and placed it in 4℃ refrigerator again. 6.For both experimental group and control group, we selected red colonies, induced by galactose for 30mins, extract the total mRNA by TRIzol. Then the mRNA was reverse transcribed by oligodT. 7.To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specfic primers. Then sequence the PCR product to proved the success of our design further.

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.