Part:BBa_K3111501

TmEncH_DARPin929_StrepII

This biobrick encodes for T. maritima encapsulin (6-His) fused to a C terminal DARPin929 with a StrepII-tag. It enabled us to determine the feasibility of construction of a multicomponent drug delivery platform and formed the intermediate building block before the manufacture of the final cytotoxic cargo loaded encapsulin based drug vehicle. Its major purpose was the study of assembly of an encapsulin monomer fused with large peptides displayed on its surface such as DARPin929. The construct is flanked by a Strep tag which allows column-based purification of the expressed protein.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 171
  Illegal BglII site found at 586
  Illegal BamHI site found at 950
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 520
  Illegal SapI.rc site found at 551
  

Experimental Results

DNA Analysis

Figure 1: Test digest of BBa_K3111201 within a pSB1C3 plasmid; a-d indicate repeats of different colonies containing the same plasmid cut with BamHI and XbaI type II restriction enzymes.

We started by cloning BBa_K3111501 into pSB1C3 vector. In order to investigate whether the ligation was successful, we picked 4 colonies from the plate containing the transformed DΗ5α, grown into 5 mL cultures, miniprepped and conducted a test digest with restriction enzymes BamHI and XbaI.

We expected bands at 2760 bp and 792 bp and those were obtained only for colony B as observed in Figure 1. Thus, the miniprepped plasmid obtained from that colony was then transformed into BL21 (DE3) into order to proceed with 50 mL cultures to express the protein.

Day 1

Different batches of E. coli BL21 (DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K3111501 sequence coding for mScarlet + DARPin fusion protein. Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. Plates were incubated at 37°C overnight.

Day 2

Transformed colonies containing pSB1C3 + BBa_K3111501 were used to prepare overnight starter cultures containing a total of 5 mL LB broth and chloramphenicol (5 μL). Cultures were incubated at 37 °C overnight.

Day 3

A 50 mL scale-up cultures was prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K3111501. The culture was incubated at 37°C until it reached an OD600 of 0.6. Once they reached OD600 0.6, the cultures were induced by addition of 400 μΜ IPTG. The cultures were left to grow again overnight at 37 °C.

Day 4

The culture was collected and transferred into a 50 mL falcon tube. It was spun for 10 minutes at 5000 rpm in order to pellet the cells. Then the supernatant was discarded and the pellet frozen at -80 °C.

Protein Analysis

Figure 2: SDS PAGE of T. maritima encapsulin monomer_DARPin929_StrepII. Purified protein is highlighted in the by the red box. M: PageRuler^TM Prestained Protein Ladder S: Cell lysate Soluble Fragment, I: Insoluble cell lysate fragment, L: Load, W: Wash, 1-3: Elution 1-3.

In order to observe whether the BBa_K3111501 fusion protein was successfully expressed, we analysed our cell pellet using SDS PAGE. The pellet obtained from the 50 mL cultures was then resuspended in Tris Buffer Saline at an OD600 of 10. Once resuspended, the sample was cell lysed using sonication. Following sonication, the sample were span to separate the soluble and insoluble fragments form the whole cell lysate. 50 μL from each sample were obtained and stained with Laemmli reagent.

We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin resin. The process involved packing the column, equilibrating the resin and loading the soluble sample. Then a washing step was performed to remove any potential non bound nonspecific proteins. Then we eluted using competitive elution by loading BXT which competed with TmEncH_DARPin_StrepII for binding sites with the resin, thus detaching the protein of interest from the column. Finally, we recycled the column ready for future purifications. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE.

The expected protein size was 51 kDa. As observed from Figure 2, a thick band at this size was observed majorly in the insoluble fragment indicating that even though expression was successful the fusion protein has considerably reduced solubility. However, after purification we managed to elute some of the expressed protein from the soluble fragment observed in elution 2. We did not observe any smaller bands, indicating that the DARPin was not cleaved off.

To observe assembly under non reducing environment, we concentrated the purified sample from elution 2 and used it for Transmission Electron Microscopy imaging and non-reducing PAGE gel.

Figure 3: a) Transmission Electron Microscopy of assembled T. maritima encapsulin_DARPin929_StrepII; Scalebar: 50 nm, b) Native PAGE gel of assembled T. maritima encapsulin_DARPin929_StrepII.

Figure 3 (a) shows the TEM image obtained. We could clearly observe assembled encapsulins thus concluding that although expression of the protein was mostly insoluble, the soluble fragment contained fully assembled encapsulins with surface displayed DARPin929. The native PAGE gel observed in Figure 3 (b) let us confirm that the DARPin did not get spliced-off during assembly, as we observed a significant increase in band size from the TmEnc lane to the TmEnc_DARPin929.

After these observations we decided that assembly was not very critically affected and moved to cargo loading in part BBa_K3111502.