Difference between revisions of "Part:BBa K2371004"

Revision as of 07:22, 31 October 2017

sgRNA generator for EML4-ALK variant A 23

This specific sgRNA generator is for detection of EML4-ALK cancer gene detection in pair with BGIC-Union NT7-dCas9(BBa_K2371000) and CT7-dCas9(BBa_K237101). This part is designed with T7 promotor and T7 terminator and can effectively generate specific RNA in vitro. The product can have RNA concentration of 1500 ng/ul or higher with MEGAscript™ T7 Transcription Kit and after the purification and collection using Ambion® mirVana™ miRNA Isolation Kit.

Figure 1. The plasmid of sgRNA generator. The sgRNA generator Biobrick is composed of a T7 promotor, coding sequence for sgRNA and a T7 terminator.

The sequence of enzyme sites, T7 promotor, sgRNA sequence and T7 terminator were synthesized by SOEPCR designed by DNAworks. The gel electrophoresis of the second amplification in SOEPCR using the oligo DNA in the start and the end of the sequence as primers validate the success of the synthesis.

Figure 2. The electrophoresis of second amplification using the using the oligo DNA in the start and the end of the sequence as primers.The desired length should be around 200bp.

Then we inserted each of those seven parts into psb1c3 plasmid.

Enzyme check and PCR(using VF and VR) check were conducted to prove the success of connection.

Figure 3. 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.

In order to synthesis RNA, we conducted a PCR to linearize the sequence. Then the purified PCR products were transcribed and purified in vitro.(Protocol ← click here)

Finally a RNA electrophoresis was conducted to corroborate the existence of RNA.

Figure 5. 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 successful obtain the sgRNA.

Sequence and Features