Difference between revisions of "Part:BBa K2455003"
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In addition; previous studies have shown proteins associated with R9, either covalently or non-covalently, to be able to enter a variety of cell types ([https://academic.oup.com/pcp/article/46/3/482/1817347 Chang <i>et al.</i>]). | In addition; previous studies have shown proteins associated with R9, either covalently or non-covalently, to be able to enter a variety of cell types ([https://academic.oup.com/pcp/article/46/3/482/1817347 Chang <i>et al.</i>]). | ||
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+ | NOGET MED USER INSERTION/CLONING | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
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Cell-Penetrating Peptides (CPPs) are small peptides, typically rich in basic residues, which are able to facilitate transport of a wide variety of cargoes across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634491/ Eudes & Chugh, 2008]). In recent years research have been done into making synthetic CPPs, especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells ([https://academic.oup.com/pcp/article/46/3/482/1817347 Chang <i>et al.</i>]). | Cell-Penetrating Peptides (CPPs) are small peptides, typically rich in basic residues, which are able to facilitate transport of a wide variety of cargoes across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634491/ Eudes & Chugh, 2008]). In recent years research have been done into making synthetic CPPs, especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells ([https://academic.oup.com/pcp/article/46/3/482/1817347 Chang <i>et al.</i>]). | ||
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+ | ==USER Cassette== | ||
+ | The biobrick carries the ??? USER Cassette which facilitates gene insertion through USER cloning. | ||
+ | |||
+ | PICTURE! | ||
+ | |||
+ | In order to insert one or more genes, the gene(s) needs the flanking regions: | ||
+ | SEQUENCES | ||
+ | |||
+ | For more information on USER cloning see ???. | ||
==Gene Insertion== | ==Gene Insertion== | ||
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===Insertion of BFP and YFP=== | ===Insertion of BFP and YFP=== | ||
+ | Smid amplification af gener samt digest/pcr af insert ind. | ||
==Cultivation, Purification and SDS-PAGE== | ==Cultivation, Purification and SDS-PAGE== |
Revision as of 07:32, 1 November 2017
Cell-Penetrating USER Cassette
This biobrick contains an AsiSI/Nb.BSMI USER Cassette with a N-terminal nona-arginine (R9) Cell-penetrating peptide which gives proteins inserted into the cassette the ability to pass through plasma membranes. In addition the USER Cassette carries a N-terminal hexa-histidine tag which allows for easy purification.
For our study we demonstrated that proteins inserted into the cassette can be purified, using the histidine-tag, and subsequently transferred into E. coli cells through the use of the R9 peptide.
In addition; previous studies have shown proteins associated with R9, either covalently or non-covalently, to be able to enter a variety of cell types (Chang et al.).
NOGET MED USER INSERTION/CLONING
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Cell-Penetrating Peptides (CPPs) are small peptides, typically rich in basic residues, which are able to facilitate transport of a wide variety of cargoes across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain (Eudes & Chugh, 2008). In recent years research have been done into making synthetic CPPs, especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells (Chang et al.).
USER Cassette
The biobrick carries the ??? USER Cassette which facilitates gene insertion through USER cloning.
PICTURE!
In order to insert one or more genes, the gene(s) needs the flanking regions: SEQUENCES
For more information on USER cloning see ???.
Gene Insertion
In order to test the ability of the biobrick to facilitate protein import in E. coli we choose to insert two different fluorescent proteins into it, SYFP2 and mTag BFP.
Opening of USER Cassette
To facilitate insertion we first had to prepare the vector, this was done in a two step digestion reaction. First to open the cassette 42 µL purified vector was mixed with 2.5 µL AsiSI, 10 µL 10x Tango Buffer, and 45.5 µL Nuclease-free water. The mixture was then incubated for 3 hours at 37 degrees Celsius. 5 µL linearised vector was run for 20 min at 100 V on a 1 % Agarose gel to check for proper linearisation.
Billede af CPP digestion
!
Evt. oven over column purification og Nb.BSMI digestion
Insertion of BFP and YFP
Smid amplification af gener samt digest/pcr af insert ind.