Difference between revisions of "Part:BBa K3506022"

Latest revision as of 04:00, 28 October 2020

Inducible double promoter system

Inducible double promoter system is composed of GAL7 promoter(BBa_K3506424) and U6 promoter(BBa_K3506021). GAL7 promoter can be induced by galactose in Cryptococcus neoformans. It is the first inducible promoter characterized in Cryptococcus neoformans. U6 promoter is used to initiate the expression of guide RNA（gRNA) in lineage tracing for eukaryotic systems. 

We put GAL7 promoter in the upstream of U6 promoter. The system can read the information of gRNA together with transcriptomic information by polyA tail.

Biology and Usage

GAL7 promoter can be induced by galactose and it is recognized by RNA polymerase II, which can add the polyA tail to its 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 to its downstream genes.

In our project, U6 promoter is used to transcribe gRNA constitutively [2], which can be recognized by Cas9. GAL7 promoter is used to transcribe the DNA of U6 promoter and gRNA at a specific time. So we can add the polyA tail to gRNA when induced, 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.

You can use our double promoter module when you need to read the information of Pol III transcription products together with transcriptomic information at a specific time. This is very significant for knowing the functions and influences of this kind of RNAs.

Sequence and Features

Design and Properties

We use U6 promoter (BBa_K3506021) to transcribe gRNA, and GAL7 promoter (BBa_K3506424) is used to transcribe the DNA of U6 promoter and gRNA when induced by galactose.

We tested the U6 promoter and GAL7 promoter system. The test is divided into two steps.

First step: to test whether GAL7 promoter will affect the production and 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 plates containing a low level of adenine, thus enabling a visual evaluation of the action of CRISPR/Cas9. Put GAL7 promoter upstream of U6 promoter only in the experimental group. Results showed that both of the two groups turned red, thus GAL7 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 specfic primers. Agarose gel electrophoresis was performed on the PCR products. There came out a correct band(Figure 2.). Then we sequenced the products and got the anticipated results.

Figure 1. A.The control group(U6 promoter-gDNA); B.The experimental group(GAL7 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(GAL7 promoter-U6 promoter-gDNA). Lane 1: Marker; Lane 2 and Lane 3: RT-PCR product of the control group(U6 promoter-gDNA); Lane 4 and Lane 5: RT-PCR product of the experimental group(GAL7 promoter-U6 promoter-gDNA) (208 bp).

Experimental approach

1. Construct recombinant plasmid. Get GAL7 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 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 Cryptococcus neoformans is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator 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 place it in an incubator at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.

6. For both the experimental group and the control group, we select pink colonies. They are induced by galactose for 30mins. Then we extract the total mRNA by TRIzol. The mRNA is reverse transcribed using oligodT as the primer.

7. To test whether gRNA can be transcribed, we perform PCR on 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. (2014). RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro. The Journal of biological chemistry, 289(29), 20396–20404.