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 their intein part further.

Large-Scale Production of Inteins

We investigated the burden of high levels of intein production in E.coli by monitoring their growth with and without the recombinant protein.

In Vivo Expression

Growth curves of BL21 (DE3) bacteria carrying an empty plasmid (pSB1C3), uninduced bacteria carrying BBa_K2842690 and induced bacteria carrying BBa_K2842690. Induction 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-inoculate into 50ml scale-up cultures and incubated at 37°C until they reached an OD 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.

Figure 2: Graph displaying the OD 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, OD was recorded for each of the samples every hour. As expected, cultures at 37 °C, displayed a higher OD since they were incubated at a temperature that allowed faster bacterial growth. Although we hypothesised that induced cultures would grow at slower rates (lower OD increase rate), this was not the case at 25°C.

Figure 3: Graph displaying the OD measurements for each of the cultures after the time of induction. Since induction was performed in control and induced cultures at 0.6 OD, the initial point for all curves (including those of non-induced samples) shown in this graph is 0.6 OD. As expected, cultures at 37 °C, displayed a higher OD since they were incubated at a temperature that allowed faster bacterial growth. Although we hypothesised that induced cultures would grow at slower rates (lower OD increase rate), this was not the case at 25°C.

In Vitro Expression

In order to maintain the modularity of our platform, we wanted to validate that it could be manufactured using cell free systems, therefore we expressed BBa_K2842690 using cell-free protein synthesis (CFPS) with bacterial cell lysate.

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