Difference between revisions of "Part:BBa K3111032"

Revision as of 20:06, 20 October 2019

Photosensitiser miniSOG fused to T. maritima encapsulin loading peptide

This part encodes E. coli optimized mini singlet oxygen generator protein (miniSOG) joined to a Thermotoga maritima encapsulin targeting peptide via (GSG)3 linker. The aim of this part is to be loaded into T. maritima encapsulin with a cell targeting peptide and illicit cytotoxic effect in cells

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 346
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 encapsulin-DARPin929 fusion proteins in constructs BBa_K3111502 and BBa_K3111503.

Protein Analysis

Just like with part BBa_K3111031 we analysed the most concentrated elutions by running an SDS PAGE (Figure 1). Lanes 3-6 all expressed TmEncH-DARPin929 fusion protein (~51 kDa), while in lanes 5 and 6 we can also observe a strong band at ~36 kDa, which corresponds to T. maritima encapsulin without DARPin929 as this construct had both TmEncH and TmEncH-DARPin929 co-expressed together.

As expected, in lanes 3, 5 and 6 we can see a fairly strong band at ~15 kDa, which corresponds to the molecular weight of BBa_K3111032. However, lane 4 does not show any miniSOG (besides the likely E. coli protein contamination), which we attribute to unsuccessful cloning as different lanes were cloned separately.

Figure 1: SDS PAGE of loaded encapsulin-DARPin929 constrcts expressed in BL21 (DE3); (1): TmEncH, (2): TmEncH-DARPin929, (3 and 4): BBa_K3111502 (5 and 6): BBa_K3111503.

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. Full protocol found <a href="https://2019.igem.org/Team:UCL/Experiments#Proteins">here</a>.

Figure 2: Quantification of sfGFP loading into T. maritima encapsulin. Error bars show 95% confidence interval.

Figure 2 shows the cargo loading capacity obtained from producing the multicomponent drug delivery platform using BBa_K3111502 and BBa_K3111503 cloning strategies. Loading capacity for BBa_K3111502 was estimated to be 13.61+-0.36 molecules of miniSOG. In contrast, BBa_K3111503 was shown to be loading 8.19+-0.21 miniSOG molecules per encapsulin. We hypothesise that this difference is caused by the surface display of DARPins slowing down the assembly of encapsulins and giving the targeting peptide present on miniSOG more time to form hydrophobic interactions to the inner shell of encapsulin monomers. Due to high error, no significant difference was shown between loading sfGFP and miniSOG, although it was hypothesised that more miniSOG molecules could be loaded due to it being noticeably smaller than sfGFP. Analysis on the methodology used to calculate the number of loaded cargo proteins can be found on BBa_K3111402 registry page.

@@ Line 33: / Line 33: @@
 ===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.
+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. Full protocol found <a href="https://2019.igem.org/Team:UCL/Experiments#Proteins">here</a>.
 [[Image:TmEncH_loading.png|500px|thumb|center|'''Figure 2:''' Quantification of sfGFP loading into T. maritima encapsulin. Error bars show 95% confidence interval.]]
 Figure 2 shows the cargo loading capacity obtained from producing the multicomponent drug delivery platform using <partinfo>BBa_K3111502</partinfo> and <partinfo>BBa_K3111503</partinfo> cloning strategies. Loading capacity for <partinfo>BBa_K3111502</partinfo> was estimated to be 13.61+-0.36 molecules of miniSOG. In contrast, <partinfo>BBa_K3111503</partinfo>  was shown to be loading 8.19+-0.21 miniSOG molecules per encapsulin. We hypothesise that this difference is caused by the surface display of DARPins slowing down the assembly of encapsulins and giving the targeting peptide present on miniSOG more time to form hydrophobic interactions to the inner shell of encapsulin monomers. Due to high error, no significant difference was shown between loading sfGFP and miniSOG, although it was hypothesised that more miniSOG molecules could be loaded due to it being noticeably smaller than sfGFP. Analysis on the methodology used to calculate the number of loaded cargo proteins can be found on <partinfo>BBa_K3111402</partinfo> registry page.