Part:BBa_K2842690

Intein Monomer 2: GFP reporter flanked with orthogonal inteins

Intein Monomer 2
Function	Create intein-spliced polymers
Use in	E. coli cells
Chassis Tested	DH5α cells
Abstraction Hierarchy	Composite Device
Related Device	BBa_K2842680
RFC standard	RFC10,RFC12,RFC21,RFC23 & RFC25 compatible
Backbone	pSB1C3
Submitted by	[http://2018.igem.org/Team:UCL UCL iGEM 2018]

This construct is designed to work with Intein Monomer 1, it is flanked with the corresponding split intein fragments for protein trans-splicing. An GFP reporter is flanked by the AcelTerL-C intein and the Npu-N intein allowing for polymerisation by protein trans splicing with other proteins flanked by 2 compatible split inteins. Like Intein Monomer 1 it has SapI cassettes to facilitate the exchange of the sequences that are flanked by inteins. This enables the polymerisation of any protein that can be synthesised.

Figure 1: BsaI digestions

(1) BsaI digested Intein Passenger BBa_K2842669
(2) BsaI digested RFP inteins BBa_K2842680
(3) BsaI digested GFP inteins BBa_K2842690
*edited to show relevant bands

Characterisation by Team UCL 2019

Team UCL 2019 was planning to use intein as part of their modular drug delivery system to join binding peptides to our delivery vesicles (encapsulins) post-expression since 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 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. As a result, we sought to characterise the 2018 UCL iGEM team’s intein part further. We investigated the burden of high levels of intein production in E.coli by monitoring cell growth with and without the recombinant protein and observed the solubility of intein hybrid proteins. Previous experiments on inteins 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).

In Vivo Expression

Growth curves of E. coli BL21 (DE3) bacteria carrying an empty plasmid (pSB1C3), uninduced bacteria carrying BBa_K2842690 and induced bacteria carrying BBa_K2842690 were evaluated. Induction with IPTG was done following the protocol listed in the protocols section, briefly: BL21 (DE3) competent cells were transformed with empty pSB1C3 plasmids and pSB1C3 plasmids containing the inserted sequence of the part BBa_K2842690. Following successful transformation, starter cultures were grown overnight, re-inoculated into 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. Figures below show the measurements that were obtained at each time after inoculation and induction. Overall, we can see that there is about 50% decrease in growth (and therefore yields) when decreasing expression temperature from 37 °C to 25 °C.

Figure 2: Graph displaying the OD600 measurements for each of the cultures after the time of inoculation. Cultures were inoculated to produce an initial concentration of 0.05, which is the initial point for all curves. After induction, which took place 155 to 205 minutes after inoculation, OD600 was recorded for each of the samples every hour. As expected, cultures at 37 °C, displayed a higher OD600 since they were incubated at a temperature that allowed faster bacterial growth. Although we hypothesised that induced cultures would grow at slower rates (lower OD600 increase rate), this was not the case at 25 °C.

Figure 3: Graph displaying the OD600 measurements for each of the cultures after the time of induction. Since induction was performed in control and induced cultures at 0.6 OD600, the initial point for all curves (including those of non-induced samples) shown in this graph is 0.6 OD600. As expected, cultures at 37 °C, displayed a higher OD600 since they were incubated at a temperature that allowed faster bacterial growth. Although we hypothesised that induced cultures would grow at slower rates (lower OD600 increase rate), this was not the case at 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 4: 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: PageRuler^TM Protein Ladder, S: Soluble cleared lysate, I: Insoluble fragment of lysate.

Figure 5: 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: PageRuler^TM Protein Ladder, S: Soluble cleared lysate, I: Insoluble fragment of lysate.

Our results suggest that lower temperature of culture growth will result in higher yield of functional GFP/intein protein (Figure 6), even though the bacterial rate of growth would drop (Figures 2 and 3). This is likely the result of the protein being more soluble after expression at 25 °C.

Figure 6: BBa_K2842690 expression under different conditions in vivo. 37: expression at 37 °C; 25: expression at 25 °C. Cultures were grown overnight, and fluorescence was measured afterwards. It can be seen that the presence of IPTG and lower incubation temperature significantly increase the amount of protein of interest synthesized at 25 °C.

In Vitro Expression

Finally, 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 8, vast majority of the protein is still insoluble (band at ~55 kDa).

Figure 8: 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1536
  Illegal BsaI.rc site found at 28
  Illegal SapI site found at 1151
  Illegal SapI.rc site found at 422