Difference between revisions of "Part:BBa K3182100"
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AsPink dropout enabling colour-screening for positive colonies. Using BamHI and SpeI or PstI on both this part assembled in pSB1C3 and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein. | AsPink dropout enabling colour-screening for positive colonies. Using BamHI and SpeI or PstI on both this part assembled in pSB1C3 and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein. | ||
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| + | The part has a very strong expression with a T7 promotor (<partinfo>BBa_I719005</partinfo>) as well as a 5'-UTR (<partinfo>BBa_K1758100</partinfo>) region which has been shown to further increase expression in E. coli (<partinfo>BBa_K1758106</partinfo>), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). This requires the T7-RNA-polymerase and bacteria with this should only be used after the C-terminal fusion protein has been assembled. Systems with the T7-RNA-polymerase can be used as well for assembly of CBD-fusion proteins, but will also express the CBD part and until it reaches a stop codon in the constitutive promoter. | ||
[[File:T--Linkoping_Sweden--pinkwhite.png|800px|thumb|center|<b>Figure 1.</b> ]] | [[File:T--Linkoping_Sweden--pinkwhite.png|800px|thumb|center|<b>Figure 1.</b> ]] | ||
Revision as of 10:32, 15 July 2019
pT7-CBDcipA-pCons-AsPink
AsPink dropout enabling colour-screening for positive colonies. Using BamHI and SpeI or PstI on both this part assembled in pSB1C3 and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein.
The part has a very strong expression with a T7 promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). This requires the T7-RNA-polymerase and bacteria with this should only be used after the C-terminal fusion protein has been assembled. Systems with the T7-RNA-polymerase can be used as well for assembly of CBD-fusion proteins, but will also express the CBD part and until it reaches a stop codon in the constitutive promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 592
Illegal NheI site found at 615 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 580
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]