Part:BBa_K1022101
pT7: RBS: His6 - SUMO: MaximinH5
This part codes for the peptide 'Maximin H5' tagged with 'His-6-SUMO' molecule. Its production is triggered by IPTG (through pT7 promoter).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 46
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 170
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 355
- 1000COMPATIBLE WITH RFC[1000]
Characterization
For more info, visit [http://2013.igem.org/Team:TU-Delft/Peptides#SUMO TU Delft iGEM13 Wiki]
Peptide production
Introduction:
In the project by iGEM 13 Team TU Delft, a SUMO-peptide fusion was opted as a suitable expression system as they make the fusion proteins more soluble. The peptide by itself is not soluble in the cytoplasm but making a fusion of peptide with Small Ubiquitin like Modifiers (SUMO) will increase the solubility of the peptide, thus increasing the cytoplasmic fraction of the peptide.
A gene was constructed in such a way that the SUMO-peptide production was driven by the strong T7 phage promoter. This gene containing plasmid was harboured in a BL21(DE3) strain that has lac promoter driven T7 polymerase. Upon induction by IPTG the SUMO peptide fusion is produced as a soluble protein fraction.
The protocol can be seen [http://2013.igem.org/Team:TU-Delft/Protocol_10#protocol_10 here].
Result:
- Colony PCR SUMO Peptide
The presence of plasmid with gene inserts encoding the SUMO-peptide (BBa_K1022101, BBa_K1022102, BBa_K1022103) was confirmed by a colony pcr. The expected size of the insert with the T7 promoter was approximately around 400 bp which was clearly evident from the agarose gel picture. An Eurogentec Smartladder MW-1700-10 (https://secure.eurogentec.com/uploads/TDS-MW-1700-10.pdf) was used to identify the size of the fragments.
Discussion:
MIC determination
The MIC of peptide on S. delphini, B. subtilis and E. coli was also done. This characterization can be seen in construct BBa_K1022110.
Also visit the [http://2013.igem.org/Team:TU-Delft/PeptideCharacterization Peptide characterization] page on TU Delft iGEM13 Wiki to know more about the MIC experiments.
n/a | pT7: RBS: His6 - SUMO: MaximinH5 |