Part:BBa_K3050000
pT7+RBS+mag-2+GFP+double_terminator
Magainin-2 expressed inside E.coli combines to cell membrane and damages it. In our project, we aimed to make BL21(DE3) lyse easier by transforming BBa_K3050000 on pSB1C3.
Usage and Biology
This is a composite part that includes T7 promotor + RBS + mag-2 + GFP + double terminators. You can use this part when you want to weaken cell membrane of E.coli,also you can visually know when it expressed. As the magainin-2 and GFP is under T7 promotor, you can easily induce it by adding IPTG. We recommend 300 μM IPTG for directly putting on colonies on plates, and 500 μM for culture solution. You want to know more what is it like when IPTG added? Read this page more or see also: https://parts.igem.org/Part:BBa_I746909:Experience
BBa_I746909 is linerized by Inverse PCR to be combined with magainin-2 sequense by In-Fusion reaction. https://parts.igem.org/Part:BBa_I746909
BL21(DE3) transformed pSB1C3 BBa_K3050000 successfully grew and emit strong green fluorescent light as shown in pictures. Thus we succeeded to prove Inverse PCR,In-Fusion reaction,and transformation went great.
GFP part is on downstream of T7 promotor and magainin-2, hence we're quite sure that this result indicates that magainin-2 is expressed at the same time.
Fig.1 BL21(DE3) pSB1C3 BBa_K3050000 expressed under visible light with negative control without IPTG induction
Fig.2 pSB1C3 BBa_K3050000 expressed under UV light with negative control without IPTG induction
Fig.3 Four biological replicates all highly expressed
Fig.4 500 μM IPTG induced BBa K3050000, strongly emitting green fluorescent light
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 190
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