Difference between revisions of "Part:BBa K4275030"

Latest revision as of 11:15, 13 October 2022

pRha-riboj

The part uses a rhamnose-inducible promoter system to achieve high level expression of GOI with the fusion of a ribozyme at the junction of the promoter and its downstream sequence.

The part is an improved version based on existing pRha part (BBa_K914003), which is characterized by its high protein expression ability. The part was later modified in the construction of pRha-riboJ-Neae-Nb to enhance expression of Nb which enables cellulosome complex to be displayed on surface of E.coli.

Usage and Biology

Ribozyme (ribonucleic acid enzymes) are RNA molecules with a variety of different functions including RNA splicing in gene expression. A synthetic self-cleaving ribozyme called RiboJ was used in this circumstance to increase the RFP expression.

RiboJ is a 75 nucleotide sequence consisted of a satellite RNA of tobacco ringspot virus derived ribozyme and a 23 nucleotide hairpin [1]. During post-transcriptional processing of mRNA, RiboJ self-cleaves to eliminate its upstream sequence and remove the promoter-associated RNA leader. Consequently, only the uncleaved hairpin sequence is presented before the RBS and gene of interest. This genetic insulation would increase gene expression level at both RNA and protein level. Gene insulation by RiboJ increases gene expression via stabilizing mRNA and reducing the degradation of the transcript, resulting in higher transcript abundance [1]. The greater mRNA abundance is then amplified by translation, elevating the production of target protein.

Characterization

The Expression of pRha-riboJ-RFP and pRha-RFP

The ability for ribozyme RiboJ to increase expression level of gene of interest downstream was tested with the composite part pRha-riboJ-RFP (Fig.1A), which is an improvement of the pRha-RFP part.

The RFP production of both pRha-riboJ-RFP group and pRha-RFP control group was quantified by fluorescence intensity and riboJ group displays significant increment in level of protein expression compared to corresponding construct without riboJ (Fig.1C and Fig.1D).

The Expression of pRha-riboJ-Neae-Nb

The results of pRha-riboJ-RFP expression successfully verified the effect of RiboJ gene insulation on increasing gene expression. We then used the part as a vector and substitute RFP sequence with coding sequence Neae-Nb (Fig.1A), which is part of the Nb-Ag3 surface display system. We cultured E.coli. BL21 with this construct, induced its expression and collected the cells. SDS-page was performed, indicating that Neae-Nb was successfully expressed (Fig.1B).

Figure 1: E.coli surface display system. (A) Construction of prha-riboJ-Neae-Nb3 and prha-riboJ-mRFP1 vectors. (B) SDS-page analysis for Neae-Nb3 expression. (C) Comparison of RFP fluorescence intensity between prha-riboJ-mRFP1 construct and prha-mRFP1 set as control. (D) Stronger positive correlation was shown between RFP fluorescence intensity and concentration of rhamnose in prha-riboJ-mRFP1 construct.

Verification of protein-protein interaction

In order to verify the natural function of Neae-Nb-Ag3 surface display system, we constructed E.coli expression vector for Ag3 domain ligated with eforRed domain to visualize the protein-protein interaction (Fig.2B). The Ag3-eforRED construct was cultured for IPTG-inducible expression. Desired proteins were identified in whole cell and supernatant samples as shown by SDS-page analysis (Fig.2C). Intact E.coli cells expressing Neae-Nb on their surface was mixed with eforRed-Ag3 supernatant. In contrast to the control group only with Neae-Nb, red fluorescence was observed in Neae-Nb-Ag3 mixture under blue-light condition, providing evidence for Nb-Ag3 interaction (Fig.2D).

Figure 2: Neae-Nb3-Ag3 surface display system expression and verification. (A) Antigen-nanobody interaction between Nb3 and Ag3 domain reported by the ligated eforRED fluorescent domain. (B) Genetic circuit construction of Ag3-eforRED vector. (C) SDS-page analysis of Ag3 expression. (D) The fluorescence indication for antigen-nanobody interaction of E. Coli surface display with an Ag3 control group and a sample group.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 91
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 91
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 91
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 91
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 91
1000
COMPATIBLE WITH RFC[1000]

References

1.Clifton, Kalen P et al. “The genetic insulator RiboJ increases expression of insulated genes.” Journal of biological engineering vol. 12 23. 29 Oct. 2018, doi:10.1186/s13036-018-0115-6

@@ Line 27: / Line 27: @@
 <h3>The Expression of pRha-riboJ-Neae-Nb</h3>
-The results of pRha-riboJ-RFP expression successfully verified the effect of RiboJ gene insulation on increasing gene expression. We then used the part as a vector and substitute RFP sequence with coding sequence Neae-Nb (Fig.1A), which is part of the Nb-Ag3 surface display system. We cultured E. coli. BL21 with this construct, induced its expression and collected the cells. SDS-page was performed, indicating that Neae-Nb was successfully expressed (Fig.1B).
+The results of pRha-riboJ-RFP expression successfully verified the effect of RiboJ gene insulation on increasing gene expression. We then used the part as a vector and substitute RFP sequence with coding sequence Neae-Nb (Fig.1A), which is part of the Nb-Ag3 surface display system. We cultured <i>E.coli</i>. BL21 with this construct, induced its expression and collected the cells. SDS-page was performed, indicating that Neae-Nb was successfully expressed (Fig.1B).
 [[Image:GreatBay SCIE--Part Fig3.png|thumbnail|750px|center|'''Figure 1:'''
-E. coli surface display system. (A) Construction of prha-riboJ-Neae-Nb3 and prha-riboJ-mRFP1 vectors. (B) SDS-page analysis for Neae-Nb3 expression. (C) Comparison of RFP fluorescence intensity between prha-riboJ-mRFP1 construct and prha-mRFP1 set as control. (D) Stronger positive correlation was shown between RFP fluorescence intensity and concentration of rhamnose in prha-riboJ-mRFP1 construct. ]]
+<i>E.coli</i> surface display system. (A) Construction of prha-riboJ-Neae-Nb3 and prha-riboJ-mRFP1 vectors. (B) SDS-page analysis for Neae-Nb3 expression. (C) Comparison of RFP fluorescence intensity between prha-riboJ-mRFP1 construct and prha-mRFP1 set as control. (D) Stronger positive correlation was shown between RFP fluorescence intensity and concentration of rhamnose in prha-riboJ-mRFP1 construct. ]]
 <h3>Verification of protein-protein interaction</h3>
-In order to verify the natural function of Neae-Nb-Ag3 surface display system, we constructed E. coli expression vector for Ag3 domain ligated with eforRed domain to visualize the protein-protein interaction (Fig.2B). The Ag3-eforRED construct was cultured for IPTG-inducible expression. Desired proteins were identified in whole cell and supernatant samples as shown by SDS-page analysis (Fig.2C). Intact E. coli cells expressing Neae-Nb on their surface was mixed with eforRed-Ag3 supernatant. In contrast to the control group only with Neae-Nb, red fluorescence was observed in Neae-Nb-Ag3 mixture under blue-light condition, providing evidence for Nb-Ag3 interaction (Fig.2D).
+In order to verify the natural function of Neae-Nb-Ag3 surface display system, we constructed <i>E.coli</i> expression vector for Ag3 domain ligated with eforRed domain to visualize the protein-protein interaction (Fig.2B). The Ag3-eforRED construct was cultured for IPTG-inducible expression. Desired proteins were identified in whole cell and supernatant samples as shown by SDS-page analysis (Fig.2C). Intact <i>E.coli</i> cells expressing Neae-Nb on their surface was mixed with eforRed-Ag3 supernatant. In contrast to the control group only with Neae-Nb, red fluorescence was observed in Neae-Nb-Ag3 mixture under blue-light condition, providing evidence for Nb-Ag3 interaction (Fig.2D).