Team:UCL/Contribution

Introduction BBa_K2842690 Experiments

Characterize: GFP/intein

Introduction

One of our main concerns about our engineered encapsulin vehicle was that the targeting peptides loaded onto the encapsulin monomers’ surface may hinder proper encapsulin assembly. We were inspired by last year’s UCL iGEM team’s work with inteins, and sought to characterise their intein part, BBa_K2842690, further. Inteins are unique proteins segments, that when joined, self-excise and join attached host proteins through a peptide bond. We thought rather than fusing the targeting peptide directly to the monomers, we could fuse the relatively small intein unit to the monomers (not hindering assembly), then subsequently add the targeting peptide with a matching intein and have it splice onto the surface of the already assembled encapsulin shells.

SETA’s 2018 Part BBa_K2842690, GFP/intein:

Last year’s UCL team created a part called Intein Monomer 2, and this is the part we chose to further characterize. It is comprised of eGFP flanked by the Npu-N intein and AceL-TerL-C intein. These are orthogonal inteins which will not splice together, but rather were designed with additional parts in mind that this part could be spliced to. Previous experiments done by Team UCL 2018 revealed that adding inteins to proteins significantly lowered their solubility, as such we aimed to prevent this by lowering expression temperature and cell-free-protein-synthesis (CFPS).

Experiments

In Vivo Expression:

First, we evaluated Growth curves of E. coli BL21 (DE3) bacteria carrying an empty plasmid (pSB1C3), uninduced bacteria carrying BBa_K2842690 and induced bacteria carrying BBa_K2842690. 50ml scale-up cultures and incubated at 37 °C until they reached an OD600 of 0.6. They were then induced with IPTG and grown at 25 °C or 37 °C. From the growth curves on the BioBrick page, we can see that there is about 50% decrease in growth (and therefore yields) when decreasing expression temperature from 37 °C to 25 °C.

Next, we ran the soluble and insoluble fractions of cell lysate on an SDS PAGE and a Western Blot using a Strep-Tactin antibody because the part has a Strep-tag. From Figures 4 and 5 we can see that the protein (highlighted by the red rectangle) expressed at 37 °C is completely insoluble, while expressed at 25 °C it is only barely soluble.

Figure 1: a) SDS PAGE gel b) Western Blot with Strep-Tactin® of the 37 °C post-induction growth scaled-up BL21* (DE3) culture. GFP/intein protein is highlighted in red. M: PageRulerTM Protein Ladder, S: Soluble cleared lysate, I: Insoluble fragment of lysate.
Figure 2: a) SDS PAGE gel b) Western Blot with Strep-Tactin® of the 25 °C post-induction growth scaled-up BL21* (DE3) culture. GFP/intein protein is highlighted in red. M: PageRulerTM Protein Ladder, S: Soluble cleared lysate, I: Insoluble fragment of lysate.

Coupled with fluorescence measurements (Figure 6 on the BioBrick page), our results suggest that lower temperature of culture growth will result in higher yield of functional GFP/intein protein, even though the bacterial rate of growth would drop. However, soluble protein titres are still likely to remain very low.

In Vitro Expression:

Next, attempting to solve the issue of solubility we decided to express GFP/intein using CFPS with bacterial cell lysate. By measuring the fluorescence of the reaction over time, we estimate to have produced 0.58±0.27 mg/L of functional GFP/intein hybrid protein, which is incredibly low for a CFPS reaction. As seen in Figure 3, majority of the protein is still insoluble (band at ~55 kDa).

Figure 3: Western Blot of GFP/intein in pSB1C3, expressed at 37 °C. M: Molecular marker; I:Insoluble fraction; S: Soluble fraction.
Conclusion

As it was not possible to express enough soluble intein protein, we decided not to use inteins in creation of our encapsulin-based drug delivery system.