Part:BBa_K3111031

Superfolder GFP fused to T. maritima encapsulin loading peptide

BBa_K2522003 modified to load into T. maritima encapsulin in order to test loading efficiency.

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 697
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental Results

This part was cloned with a T7 promoter and a strong RBS to express along with T. maritima encapsulins in the same plasmid (BBa_K3111401 and BBa_K3111402) or co-transformed in a low copy count plasmid (pBAD30).

Protein Analysis

We analysed the purification of BBa_K3111401 and BBa_K3111402 cell lysates in Figure 1. These images suggest that there is a significant increase in the amount of sfGFP in the elution fractions for the encapsulin with 6-His tag insert.

Figure 1: a) SDS PAGE of BBa_K3111401 expressed in BL21 (DE3); b) SDS PAGE of BBa_K3111402 expressed in BL21 (DE3); L: PageRuler Protein Ladder M: PageRuler Plus Protein Ladder, S: Soluble cleared lysate, I: Insoluble fragment of lysate, W: Wash, L: Load, E1-3: Elutions 1-3.

We then tested a couple more loading configurations in Figure 2, including co-expressing sfGFP and encapsulin on different plasmids, as well as a sfGFP control without a targeting peptide (BBa_K2522003). The gel indicated that the only construct that showed significant loading was BBa_K3111402. In addition, it was hard to determine loading efficiency from the gel as even the control elutions (1 and 4) seemed to show faint bands near the size of sfGFP protein (28 kDa). As a result, lanes with unloaded and loaded constructs seemed to look similar. To further analyse the difference we used GelAnalyser software in Figure 3. This showed that only lane 5 (containing BBa_K3111402) had a significant peak at the sfGFP.

Figure 2: SDS PAGE of elutions from different constructs showing encapsulation of sfGFP; L: PageRuler Protein Ladder, (1): TmEnc control, (2): TmEnc + sfGFP, (3): TmEnc + sfGFP in pBAD30, (4): TmEncH control (5): TmEncH + sfGFP (6): TmEncH + sfGFP (no tag), (7): TmEncH + sfGFP in pBAD30.

Figure 3: Analysis of lanes 4-6 from the gel in Figure 2 using GelAnalyzer 2010a. Note that MW estimations are inaccurate due to the image being slightly tilted.

To get a better quantification we followed by doing spectrophotometric analysis on these elutions.

Quantification of Cargo Loading

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 autofluorescence of encapsulins. 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.

Figure 4: Quantification of sfGFP loading into T. maritima encapsulin. Error bars show 95% confidence interval.

From Figure 4, we can see that BBa_K3111402 loaded well compared to other configurations tested, with around 14.5 sfGFP molecules per encapsulin. A high variance was observed in this sample, which we deemed is a result of aggregation after discussing it with our supervisors. Alternative version of the sequence that contained sfGFP without the T. maritima encapsulin targeting peptide 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_K3111401containing 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.