Regulatory
Part:BBa_K1955001
Designed by: Justine Hsu Group: iGEM16_CGU_Taiwan (2016-10-14)
Leish pSB1C3-2.3intron
The Leish-2.3intron is an intrinsic sequence between two highly expressed coding regions, p36 and NAGT, that contains stage-independent splicing sites in Leishmania. In Leishmania, the polycistronic RNA will be spliced into two transcripts and then be translated into two proteins. The proteins will thus be constitutively expressed. We designed this biobrick in order to enhance the expression of both the drug resistant gene and the target protein.
This biobrick needs to be used with the Leish-5'UTR-HYG and Leish-3'UTR that we provide as a complete protein expression system for Leishamnia. Add the protein sequence in between the Leish-2.3 intron and Leish-3'UTR, and then add Leish-5'UTR-HYG before all the parts for Leishmania to expression your target protein stage-independently.
(1) Construction of pSB1C3-2300 intron:
Since the 2300 bp intrinsic sequence contained too many CG pairs, it couldn’t be synthesized. We used point mutation to change the nucleotide in the 2300 bp sequence, therefore, the sequence would be separated into 3 parts, the first and the second part were about 400~450 bp and the third part was approximately 1500 bp in length. Through the PCR, we could have these 3 parts amplified from p6.5 plasmid. We used the PCR-after-ligation strategy, ligating the first and second part together and performed PCR to amplify the sequence. Next, ligated the part 1 +part 2 sequence with part 3, and amplify the ligated parts with PCR again. The reason why we used the PCR-after-ligation strategy was because the ligation rate of the sequence was really low. However, although the parts of 2300 intron could be proliferated by PCR, we were unable to ligate the 3 parts together. The sequencing results of the 2300 intron always lost the second part, no matter what strategy we used in the construction. So, it turned out that we couldn’t put the 2300 intrinsic region into the final construction of our shuttle vector.
All the parts of 2300 intron checked by PCR:
The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. Lane A to lane C were the three parts of 2300 intron, the first part was 400 bp, the second part was about 450 bp, and the third part was 1500 bp. Lane D was the ligation of part 1 + part 2, which would be approximately 800 bp. Lane E was the ligation of all three parts, which would be 2.3 kb in length. However, the second part would always be lost during the construction.
All the parts of 2300 intron checked by PCR:
The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. Lane A to lane C were the three parts of 2300 intron, the first part was 400 bp, the second part was about 450 bp, and the third part was 1500 bp. Lane D was the ligation of part 1 + part 2, which would be approximately 800 bp. Lane E was the ligation of all three parts, which would be 2.3 kb in length. However, the second part would always be lost during the construction.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1963
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 989
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Categories
Parameters
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