Part:BBa_K3111402
TmEncH_StrepII + sfGFP_TP
T. maritima encapsulin containing 6-His insert co-expressed with sfGFP in order to measure loading capacity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 183
Illegal BglII site found at 598
Illegal BamHI site found at 1993 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1013
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1218
Illegal BsaI.rc site found at 2213
Illegal SapI.rc site found at 532
Illegal SapI.rc site found at 563
Experimental Results
To measure encapsulin loading, spectrophotometric analysis of purified sample was performed. The sample was excited at 280 nm and 485 nm, finding total protein concentration and fluorescent intensity of the sample. Then, a calibration curve was used to determine sfGFP concentration, adjusting for interference. Finally, this concentration was subtracted from the total protein concentration, in order to get encapsulin concentration. Concentrations were converted to molarity, and a ratio between encapsulins and cargo molecules was calculated.
From Figure 1, we can see that BBa_K3111402 loaded well compared to other configurations tested, with around 14.5 sfGFP molecules per encapsulin. Alternative version of the sequence that contained BBa_K2522003 instead of BBa_K3111031 showed minimal loading (likely resulting only due to impurities in the sample), demonstrating that the targeting peptide sequence attached to BBa_K3111031 improve the part by letting it load into T. maritima encapsulin.
Part BBa_K3111401 containing T.maritima encapsulin without 6-His insert was also tested and shown to load only about 2 sfGFP molecules per encapsulin. Therefore, we chose to use BBa_K3111003 over BBa_K3111002 as the building block of our delivery system due to increased loading efficiency.
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