iGEM 2017 — Team BGIC-Union

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# Team:BGIC-Union/Notebook

Team：BGIC-Union

[BGIC-Union](https://2017.igem.org/Team%3ABGIC-Union)

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# Team:BGIC-Union/Experiments

Team：BGIC-Union

[BGIC-Union](https://2017.igem.org/Team%3ABGIC-Union)

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# Team:BGIC-Union/Results

Team：BGIC-Union

[BGIC-Union](https://2017.igem.org/Team%3ABGIC-Union)

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* [Safety](https://2017.igem.org/Team%3ABGIC-Union/Safety)
* [EVALUATION](https://2017.igem.org/Team%3ABGIC-Union/Evaluation)

## 1. Expression of PC report system

Construct expression system for T7 PC report system and Fluc PC report system.

T7 PC report system:
![](https://static.igem.org/mediawiki/2017/1/17/Pro8.jpg )

After restrictive enzyme digestion, all of the seven sgRNA were inserted into psb1c3 plasmid. All of the plasmids were then transformed in to Eco.li strain DH5a and amplified. Sequencing of the samples was done by BGI and most of them were successful.
We also conducted restrictive enzyme check and PCR check to determine its accuracy.
![](https://static.igem.org/mediawiki/2017/2/22/BGIC-Union_Part_sgRNA_Figure3.png)

Figure 12. PCR check of seven sgRNA generators. The PCR check is conducting using VF and VR as primers. Marker is noted as M which has a ladder of 200bp. Control is a J23119 biobrick and noted as C. PCR result of control should be about 350bp. VA23 stands for sgRNA generator for EML4-ALK Variant A 23 and so on. The length of the PCR result of sgRNA generator should be about 500bp.

The in vitro transcription and purification of sgRNA.
We used MEGAscript™ T7 Transcription Kit to yield sgRNA in vitro.
A PCR was conducted to obtain the linear T7 promotor – sgRNA – T7 terminator cassette. The PCR product was then mixed with T7 RNA polymerase, ribonucleotides and reaction buffer.
After six hours of incubation at 37 degrees centigrade, DNase was introduced to remove the template DNA.
We used a commercial miRNA purification kit to separate the transcribed sgRNA.(Ambion® mirVana™ miRNA Isolation Kit)
After turns of wash and purification, the final RNA product diluted in nuclease-free water can reach the concentration of 1500 ng/ul or higher.
Also, a RNA electrophoresis was conducted to examine the existence of sgRNA.
![](https://static.igem.org/mediawiki/2017/8/85/BGIC-Union_Part_sgRNA_Figure5.png)

Figure 13. Native RNA electrophoresis of all the seven sgRNA. Because our RNA electrophoresis is native electrophoresis, the each RNA won’t concentrate to a band since they may form higher structure. The only purpose we did it was to ensure we had successfully obtain the sgRNA.

The sgRNA samples were then stored in -20 degrees centigrade.

### 3. NASBA

Nucleic acid sequence based amplification (NASBA) is a method in molecular biology that is used to amplify RNA sequences under a constant temperature. We decide to add this procedure to counter with the extremely low concentration of ctDNA in blood. We believe it is better than PCR because 1) Based on our modeling, too much target DNA concentration will reduce the efficiency of the system. 2) it is easier to conduct and does not require experimental machine.
We conducted NASBA with a gradient of diluted Target DNA to demonstrate the success of NASBA.
Procedures:
1. Dilute the target sequence(both Variant A and Variant B) in to 15nM、1.5nM、0.15nM、15pM、1.5pM、0.15pM.
2. Mix T7 polymerase buffer, AMV RT buffer, rNTPs, dNTPs, DEPC water and T7 polymerase together.
3. Process the diluted DNA in 65 degrees centigrade for 5mins and then retrieve to 41 degree centigrade.
4. Mix the NASBA system with diluted DNA.
5. Gel electrophoresis.
We compared the PCR result with NASBA result.

![](https://static.igem.org/mediawiki/2017/9/9b/BGICVARIANTA.jpg)

Figure 14. Comparison between PCR result and NASBA result with a gradient of initial concentrations(Variant A). The initial concentrations are 15nM, 1.5nM, 0.15Nm, 15pM, 1.5pM and 0.15pM.

![](https://static.igem.org/mediawiki/2017/b/bc/BGICVARIANTVB.jpg)

Figure 15. Comparison between PCR result and NASBA result with a gradient of initial concentrations(Variant B). The initial concentrations are 15nM, 1.5nM, 0.15Nm, 15pM, 1.5pM and 0.15pM.

As the figures show, we have successfully amplified the target sequence.

## [Parts](https://2017.igem.org/Team%3ABGIC-Union/Parts)/[Lab Journal](https://2017.igem.org/Team%3ABGIC-Union/Lab)