Difference between revisions of "Part:BBa K2524011:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | To improve our previous Mambalgin-1 part | + | <b>To improve our previous Mambalgin-1 part</b> |
We improved our previous Mambalgin-1 construct (https://parts.igem.org/Part:BBa_K1110003) for E. coli this year by removing extra base pairs that coded for a portion of the signal peptide segment. The new sequence produces the Mambalgin-1 protein without any signal peptide. Next, we added a TAC promoter, LAC operon, GST-Tag, and an HRV-3 protease site upstream of the Mambalgin-1 coding sequence. This increased the size of our target protein by 26 kDa, which made protein band detection much easier on SDS PAGE. Subsequently, the strategy solved our issues related to losing our protein band in the SDS PAGE dye-front. | We improved our previous Mambalgin-1 construct (https://parts.igem.org/Part:BBa_K1110003) for E. coli this year by removing extra base pairs that coded for a portion of the signal peptide segment. The new sequence produces the Mambalgin-1 protein without any signal peptide. Next, we added a TAC promoter, LAC operon, GST-Tag, and an HRV-3 protease site upstream of the Mambalgin-1 coding sequence. This increased the size of our target protein by 26 kDa, which made protein band detection much easier on SDS PAGE. Subsequently, the strategy solved our issues related to losing our protein band in the SDS PAGE dye-front. | ||
The GST tag also allowed for affinity gravity column chromatography purification. In fact, the HRV-3 protease site made cleavage of Mambalgin-1 from the GST tag possible in our lab (we don’t have access to FPLC). | The GST tag also allowed for affinity gravity column chromatography purification. In fact, the HRV-3 protease site made cleavage of Mambalgin-1 from the GST tag possible in our lab (we don’t have access to FPLC). | ||
| − | + | <html> | |
Uniprot research of Mambalgin-1 protein conducted: | Uniprot research of Mambalgin-1 protein conducted: | ||
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- Original protein sequence contains an initial signal sequence which was not included | - Original protein sequence contains an initial signal sequence which was not included | ||
| − | The construct was submitted to IDT in minigene format. | + | <b>The construct was submitted to IDT in minigene format.</b> |
| + | <ul><b>Results</b>:<table><tr><td><img width="75%" height="auto" src="https://static.igem.org/mediawiki/2017/6/69/T--Georgia_State--Mamba_IDT.png"></td></tr></table> | ||
| − | Primers were designed to add restriction sites in order to remove the sequence from the plasmid. | + | |
| + | |||
| + | <b>Primers were designed to add restriction sites in order to remove the sequence from the plasmid.</b> | ||
Mamba EcoRI Forward | Mamba EcoRI Forward | ||
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| − | PCR to create official biobrick prefix site proximal to Tac promoter | + | <b>PCR to create official biobrick prefix site proximal to Tac promoter</b> |
Mamba Add XbaI Forward | Mamba Add XbaI Forward | ||
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| − | Mutagenesis to remove second EcoRI site upstream of Mamba CDS | + | <b>Mutagenesis to remove second EcoRI site upstream of Mamba CDS</b> |
EcoRI Mutagen Forward-sense | EcoRI Mutagen Forward-sense | ||
| Line 55: | Line 58: | ||
| − | Protein Expression | + | <b>Protein Expression</b> |
Protein purification - GST sepharose 4B resin | Protein purification - GST sepharose 4B resin | ||
| − | Column protease cleavage with HRV-3 protease -> elute pure Mambalgin-1 | + | Column protease cleavage with HRV-3 protease -> elute pure Mambalgin-1 afterward |
Protein Detection - western blot with monoclonal Anti-GST antibodies | Protein Detection - western blot with monoclonal Anti-GST antibodies | ||
| − | + | f | |
===Source=== | ===Source=== | ||
Latest revision as of 03:58, 2 November 2017
Mambalgin-1 in E. coli Improvement
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 763
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 160
Design Notes
To improve our previous Mambalgin-1 part
We improved our previous Mambalgin-1 construct (https://parts.igem.org/Part:BBa_K1110003) for E. coli this year by removing extra base pairs that coded for a portion of the signal peptide segment. The new sequence produces the Mambalgin-1 protein without any signal peptide. Next, we added a TAC promoter, LAC operon, GST-Tag, and an HRV-3 protease site upstream of the Mambalgin-1 coding sequence. This increased the size of our target protein by 26 kDa, which made protein band detection much easier on SDS PAGE. Subsequently, the strategy solved our issues related to losing our protein band in the SDS PAGE dye-front.
The GST tag also allowed for affinity gravity column chromatography purification. In fact, the HRV-3 protease site made cleavage of Mambalgin-1 from the GST tag possible in our lab (we don’t have access to FPLC). Uniprot research of Mambalgin-1 protein conducted: - Determined functional portion of protein sequence --> trim down unnecessary amino acids - Original protein sequence contains an initial signal sequence which was not included The construct was submitted to IDT in minigene format.
- Results:
 |