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Team:Hannover/Protocols/Cloning/Parts
From 2014.igem.org
Results / Cloning the Shipping Vectors
We wanted to contribute useful Biobricks to the iGEM Parts Registry and thus cloned our important constructs into the iGEM pSB1C3 standard vector. In accordance with classical cloning, we amplified our sequences of interest, cleaved them with restriction endonucleases generating the same single-stranded overhangs as for the final vector backbone and ligated both parts finally. After the ligation products were transferred into E.coli and analyzed via colony PCR, the resulted plasmids were isolated and sent to the iGEM Headquater.
Labwork
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Figure 1: Immunostaining of Nicotiana tabacum leave extracts after infiltrating them with pORE-E3_2x35S_T4MBP-containing Rhizobium radiobacter solution (T4MBP). Due to a failed immunologic detection, no difference between the samples infiltrated by water (control) and by bacteria solution were observed (1A). Another repetition A final volume of 15 µl of leave extracts was separated via 12 % SDS-PAGE, proteins were transferred onto a PVDF membrane and decorated by an anti-FLAG-tag antibody. Black arrows indicate the expected molecular weight of the T4MBP. The standard (M) used here is the Spectra Multicolor Broad Range Protein Ladder. |
As bacterial host for the heterologous T4MBP production we chose Origami2 cells to work with. This E. coli strain expresses huge amounts of cytosolic disulfide isomerase and thus raises the disulfide bond formation for recombinant proteins. Furthermore, to improve the quality of proteins, we lowered the expression temperatures to 16 °C.
E.coli based Expression
- Cloning the metal-binding-sequences into the pASK plasmid
- Heterologous Expression of T4MBP.
- Lysis of bacteria cells and protein precipitation by TCA.
- Analysis by SDS-PAGE.
- Transfer of separated proteins onto a PVDF membrane and an immunostaining by an anti-6xHistidine-tag antibody.
Results
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Figure 2: Immunostaining of cytosolic extracts from pASK_T4MBP containing Origami2TM cells (T4MBP). While the control (c) shows no reaction, the T4MBP medium elucidated a specific signal at a molecular weight of ~40 kDa. A final volume of 15 µl of prepared proteins was separated by 12 % SDS-PAGE. Proteins were than transferred onto a PVDF membrane and decorated by an anti-6xHistidine-tag antibody. A black arrow indicates the expected molecular weight. The standard (M) used here was the Spectra Multicolor Broad Range Protein Ladder. |
