Part:BBa_K5269023

SpyTag-Mgfp-5-His-Tag and BsmB I cut site(for GGA)

It contains SD-sequences (BBa_K5269034), SpyTag, Mgfp-5 (BBa_K5269013), and His-Tag sequences. In addition, the BsmB I cleavage sites at both ends of the sequence allow for Golden Gate Assembly to the Golden Gate Assembly Site (BBa_K5269020) and T7 promoter-shRNA-T7 terminator-Golden Gate Assembly Site (BBa_K5269025). The SpyCatcher-SpyTag system works when used with Lpp-OmpA-SpyCatcher (BBa_K5269026) or INPNC-SpyCatcher (BBa_K5269027). Therefore, we expect the adhesion protein Mgfp-5 to be stable on the Membrane Vesicles, which will facilitate the retention of our shRNA-encapsulated membrane vesicles on the plant surface.

Here are the results of SDS-PAGE of OmpA-Mgfp5 and INP-Mgfp5 bound by SpyCatcher-SpyTag. See our results page (https://2024.igem.wiki/tokyotech/results/) for details.

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Serial	Stock Name	Scaffolding Protein - Display Protein	Molecular Weight (kDa)	Corresponding SDS-PAGE Lanes (Periplasmic/Cytoplasmic/Membrane Fractions)	Presence of Bands (Periplasmic/Cytoplasmic/Membrane Fractions)
O_1	O_Mgfp5	OmpA-Mgfp5	36.8	1/2/3	Yes/Yes/Yes
I_1	I_Mgfp5	INP-Mgfp5	54.2	4/5/6	Yes/Yes/Yes
O_2	O_Glucanases	OmpA-Glucanases	78.5	7/8/9	Yes/Yes/Yes
I_2	I_Glucanases	INP-Glucanases	95.9	10/11/12	Yes/Yes/Yes
O_3	O_Mgfp5-Glucanases	OmpA-Glucanases, OmpA-Mgfp5	78.5, 36.8	13/14/15	No/Yes/Yes, Yes/Yes/Yes
I_3	I_Mgfp5-Glucanases	INP-Glucanases, INP-Mgfp5	95.9, 54.2	16/17/18	No/No/No, No/Yes/Yes
O_4	O_Glucanases-GH19Chitinase	OmpA-Glucanases, OmpA-GH19Chitinase	78.5, 56.5	19/20/21	No/Yes/Yes, No/No/No
I_4	I_Glucanases-GH19Chitinase	INP-Glucanases, INP-GH19Chitinase	95.9, 73.9	22/23/24	Yes/Yes/No, No/No/No

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 381
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]