Composite
Part:BBa_K1955007
Designed by: Chia-Wei Chang Group: iGEM16_CGU_Taiwan (2016-10-16)
pSB1C3-5'HYG-GFP-3'UTR
A shuttle vector is a vector constructed so that it can reproduce in two different host species. The vector can be quickly amplified in E. coli and then manipulated in Leishmania. This shuttle vector consist of Hygromycin resistant gene as drug selection marker and GFP for easy validation of transfection. It is useful in trying the condition of electroporation of Leishmania since the GFP is easy to observe.
(1) Construction of pSB1C3-5’HYG-GFP-3’UTR:
Since we can’t detect the HA and OVA protein by western blotting after the pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR plasmid were transfected into leishmania. We decided to construct pSB1C3-5’HYG-GFP-3’UTR in order to prove if our leishmania shuttle vector could express the second protein or not. The GFP sequence came from BBa_E0040 in the vector pSB1A2.
The pSB1C3-3’UTR was digested with EcoRI and XbaI, The pSB1A2-GFP and pSB1C3-5’HYG were digested with EcoRI and SpeI. After the purification, the pSB1C3-3’UTR was ligated with GFP and 5’HYG successively, then transformed into DH5a. The colonies were checked by colony PCR. The right length of GFP-3’UTR should be approximately 1.5 kb (720 bp +774 bp), while the 5’HYG-GFP-3’UTR should be about 3 kb (1446 bp +720 bp +774 bp). As the result, we knew that all the colonies contained the correct plasmid after the construction. The right colony of pSB1C3-5’HYG-GFP-3’UTR was picked and amplified in 200 ml LB broth, then the plasmid DNA was purified by midiprep.
pSB1C3-GFP-3’UTR and pSB1C3-5’HYG-GFP-3’UTR check by colony PCR
The PCR was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The GFP-3’UTR was about 1.5 kb in length, and the 5’HYG-GFP-3’UTR was about 3 kb.
The pSB1C3-3’UTR was digested with EcoRI and XbaI, The pSB1A2-GFP and pSB1C3-5’HYG were digested with EcoRI and SpeI. After the purification, the pSB1C3-3’UTR was ligated with GFP and 5’HYG successively, then transformed into DH5a. The colonies were checked by colony PCR. The right length of GFP-3’UTR should be approximately 1.5 kb (720 bp +774 bp), while the 5’HYG-GFP-3’UTR should be about 3 kb (1446 bp +720 bp +774 bp). As the result, we knew that all the colonies contained the correct plasmid after the construction. The right colony of pSB1C3-5’HYG-GFP-3’UTR was picked and amplified in 200 ml LB broth, then the plasmid DNA was purified by midiprep.
pSB1C3-GFP-3’UTR and pSB1C3-5’HYG-GFP-3’UTR check by colony PCR
The PCR was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The GFP-3’UTR was about 1.5 kb in length, and the 5’HYG-GFP-3’UTR was about 3 kb.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2096
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Categories
Parameters
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