Difference between revisions of "Part:BBa K3506095"

Revision as of 13:47, 24 October 2020

Constitutive double promoter module

Constitutive double promoter system is composed of GAPDH promoter (BBa_K3506020) and CnU6 promoter(BBa_K3506021) . It is generally accepted that GAPDH promoter (pGAP) is considered to be a strong constitutive promoter which is used by RNA polymerase II. U6 promoter(pU6) is used to drive the expression of homing guide RNA（hgRNA） in lineage tracing for eukaryotic systems. We put pGAP in the upstream of pU6. The system can read the information of snRNAs out of the transcriptomic information by polyA tail.

Biology and Usage

pGAP is considered to be a strong constitutive promoter which is promoted by RNA polymerase II. 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. 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. It is known that RNA polymerase III transcription product does not have polyA and cannot be captured by OligodT 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.

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 pU6 and pGAP systems. The test is divided into two steps. First step: to test whether pGAP will affect the production and the function of gRNA. We put gRNA targeting ADE2 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. Second step: to test that whether gRNA can be reverse transcribed using oligodT 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 oligodT 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.

Experimental approach

1.Construct recombinant plasmid. Get pGAP from the genome of Cryptococcus neoformans. Insert it upstream of pU6 on PRH003 plasmid. 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. 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 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.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3506095 short</partinfo>
-Constitutive double promoter system is composed of GAPDH promoter(BBa_K3506020) and CnU6 promoter(BBa_K3506021).
+Constitutive double promoter system is composed of GAPDH promoter (BBa_K3506020) and CnU6 promoter(BBa_K3506021) .
+It is generally accepted that GAPDH promoter (pGAP) is considered to be a strong constitutive promoter which is used by RNA polymerase II.
-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.
 U6 promoter(pU6) is used to drive the expression of homing guide RNA（hgRNA） in lineage tracing for eukaryotic systems.
+We put pGAP in the upstream of pU6. The system can read the information of snRNAs out of the transcriptomic information by polyA tail.
-We put pGAP in the upstream of pU6. The system can read the information of snRNAs out of transcriptomic information by polyA tail.
 <b><font size"3">Biology and Usage</font></b>
-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 pGAP to drive the transcription of RNA polymerase III promoter of the U6 small RNA gene and the genes downstream of pU6.
+pGAP is considered to be a strong constitutive promoter which is promoted by RNA polymerase II.
+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.
+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.
+It is known that RNA polymerase III transcription product does not have polyA and cannot be captured by OligodT 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.
 <!-- -->
@@ Line 26: / Line 24: @@
 <b><font size"3">Properties</font></b>
-We tested the pU6 and pGAP system. The test is divided into two parts.
+We tested the pU6 and pGAP systems. The test is divided into two steps.
+First step: to test whether pGAP will affect the production and the function of gRNA. We put gRNA targeting ADE2 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 part: to test whether pGAP will affect the production and function of gRNA. We put sgRNA which targets ADE2 gene downstream of pU6 in both experimental group and control group. Put pGAP upstream of pU6 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)
+Second step: to test that whether gRNA can be reverse transcribed using oligodT 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 oligodT 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.
-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 is reverse transcribed by oligodT. To test whether gRNA can be transcribed, we perform PCR on reverse transcription products by two specfic primers. Agarose gel electrophoresis is performed on the PCR product. There come out a correct band(图）. Then we sequence the products and get the right result.
 <b><font size"3">Experimental approach</font></b>
-.Construct recombinant plasmid. Get pGAP from the genome of Cryptococcus neoformans. Inserted it upstream of pU6 on PRH003 plasmid.
+.Construct recombinant plasmid. Get pGAP from the genome of Cryptococcus neoformans. Insert it upstream of pU6 on PRH003 plasmid.
+.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.
-.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.
+.Use Kpn1 enzyme to linearise the plasmids and transform them into Cryptococcus neoformans by electroporation.
+.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℃.
-.Use Kpn1 enzyme to linearise the plasmid and transformed it into Cryptococcus neoformans by electroporation.
+.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.
+.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.
-.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.
+.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.
-.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.
-.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 pGAP). These proved that pGAP won't influence the original function of pU6 and gRNA.
 <b><font size"3">References</font></b>