Difference between revisions of "Part:BBa K4620014"
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LNSQLLTWR is a mutant of LNSQLLVWR, a naturally occuring peptide formed after tryptic digestion of CcaBURP2 protein. Wild type protein contains core peptide fragment QLLVW. | LNSQLLTWR is a mutant of LNSQLLVWR, a naturally occuring peptide formed after tryptic digestion of CcaBURP2 protein. Wild type protein contains core peptide fragment QLLVW. | ||
| − | QLLVW is a peptide that can be cyclised by BURP domain protein from eastern redbud (Cercis canadensis ( | + | QLLVW is a peptide that can be cyclised by BURP domain protein from eastern redbud (Cercis canadensis (Part code), forming stephanotic acid. This side chain macrocyclisation reaction is catalysed by copper (II) ions. Cyclisation happens between the Cβ of leucine and indole cycle 6- position of tryptophan residue. As of this day, mechanism of this reaction has not been fully characterised. |
| + | https://static.igem.wiki/teams/4620/wiki/att-ls20.png | ||
In the nature, LNSQLLVWR is conjugated to the N-terminal domain of Cca-BURP. iGEM 2023 team Latvia-Riga produced this peptide separately in order to make trans-cyclisation reaction where the cyclisation substrate is not attached to the catalytic domain. We chose substrate a little bit longer than the core domain in order to make expression and purification easier. | In the nature, LNSQLLVWR is conjugated to the N-terminal domain of Cca-BURP. iGEM 2023 team Latvia-Riga produced this peptide separately in order to make trans-cyclisation reaction where the cyclisation substrate is not attached to the catalytic domain. We chose substrate a little bit longer than the core domain in order to make expression and purification easier. | ||
We designed several modificated core peptide analogues in order to see what are the limits of CcaBURP2 cyclisation capacity. In this peptide valine is replaced with threonine. | We designed several modificated core peptide analogues in order to see what are the limits of CcaBURP2 cyclisation capacity. In this peptide valine is replaced with threonine. | ||
| − | |||
===Cloning=== | ===Cloning=== | ||
Our team cloned LNSQLLTWR gene into pEXP-GB1 expression vector, containing GB1 (domain B1 of Immunoglobulin G-binding protein G from Streptococcus sp.), N-terminal 8xHis tag and TEV protease cleavage site. | Our team cloned LNSQLLTWR gene into pEXP-GB1 expression vector, containing GB1 (domain B1 of Immunoglobulin G-binding protein G from Streptococcus sp.), N-terminal 8xHis tag and TEV protease cleavage site. | ||
| + | https://static.igem.wiki/teams/4620/wiki/att-ls21.png | ||
| + | https://static.igem.wiki/teams/4620/wiki/att-ls22.png | ||
| + | |||
| + | LNSQLLVWR insert amplification using PCR; positive clone checking with PCR | ||
===Expression=== | ===Expression=== | ||
Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. Expression tests were carried out to determine best expression conditions. | Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. Expression tests were carried out to determine best expression conditions. | ||
| + | |||
| + | https://static.igem.wiki/teams/4620/wiki/att-ls23.png | ||
Samples with highest expression yields were then subjected to solubility testing using BugBuster kit. Cells were lysed, samples (T = total) were taken. Lysate was centrifugated and supernatant (S = soluble) samples were taken. It can be seen that despite the fusion tag, most of the expressed protein is insoluble. | Samples with highest expression yields were then subjected to solubility testing using BugBuster kit. Cells were lysed, samples (T = total) were taken. Lysate was centrifugated and supernatant (S = soluble) samples were taken. It can be seen that despite the fusion tag, most of the expressed protein is insoluble. | ||
| + | |||
| + | https://static.igem.wiki/teams/4620/wiki/att-ls24.png | ||
| + | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 15:57, 12 October 2023
CcaBURP2 tryptic peptide V4T mutant containing the core peptide
LNSQLLTWR is a mutant of LNSQLLVWR, a naturally occuring peptide formed after tryptic digestion of CcaBURP2 protein. Wild type protein contains core peptide fragment QLLVW. QLLVW is a peptide that can be cyclised by BURP domain protein from eastern redbud (Cercis canadensis (Part code), forming stephanotic acid. This side chain macrocyclisation reaction is catalysed by copper (II) ions. Cyclisation happens between the Cβ of leucine and indole cycle 6- position of tryptophan residue. As of this day, mechanism of this reaction has not been fully characterised.
In the nature, LNSQLLVWR is conjugated to the N-terminal domain of Cca-BURP. iGEM 2023 team Latvia-Riga produced this peptide separately in order to make trans-cyclisation reaction where the cyclisation substrate is not attached to the catalytic domain. We chose substrate a little bit longer than the core domain in order to make expression and purification easier. We designed several modificated core peptide analogues in order to see what are the limits of CcaBURP2 cyclisation capacity. In this peptide valine is replaced with threonine.
Cloning
Our team cloned LNSQLLTWR gene into pEXP-GB1 expression vector, containing GB1 (domain B1 of Immunoglobulin G-binding protein G from Streptococcus sp.), N-terminal 8xHis tag and TEV protease cleavage site.
LNSQLLVWR insert amplification using PCR; positive clone checking with PCR
Expression
Obtained plasmid was transformed into T7 Express competent E. coli cells and plated on LB agar plate containing ampicillin. Expression tests were carried out to determine best expression conditions.
Samples with highest expression yields were then subjected to solubility testing using BugBuster kit. Cells were lysed, samples (T = total) were taken. Lysate was centrifugated and supernatant (S = soluble) samples were taken. It can be seen that despite the fusion tag, most of the expressed protein is insoluble.
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]