Parts

“The whole is more than the sum of its parts” - Aristotle

All parts used for IBDetection to be successful can be found on these pages. Part Overview gives a brief overview of all parts used in the sensor-reporter system and the Part Collection explains our used and designed collection in more detail, which is separated in the Basic Parts page and the Composite Parts page. Furthermore, the button Improvement brings you to the page of our improved part.

Part Overview Part Collection Basic Parts Composite Parts Improvement

Improvement

RFP coding device containing translation enhancing 5’UTR (BBa_K3972005)

The expression of fluorescent proteins can be used for a great range of biological applications [1]. To test the influence of important components of gene fragments on protein expression, e.g. promoters or ribosome binding sites, fluorescent proteins can be expressed and characterized. The development of Red Fluorescent Protein (RFP) was motivated by the discovery that living cells are less sensitive to longer wavelength excitation and under these conditions the decrease of autophosphorylation. Therefore, the emission range of fluorescent proteins should be extended [1]. Monomeric RFP1 (mRFP1) is reproduced from Discosoma striata (DsRed) and is often used to visualize the location of a protein, to observe physiological processes, and to distinguish transgenic expression in vivo [1,2]. Fluorescence absorbance of mRFP1 can be measured at an emission of 607 nm, under excitation of 584 nm [2].

RFP coding device with RBS (BBa_K801100)

Part BBa_K801100 is an RFP coding device, which is RFC[10] and RFC[25] BioBrick compatible and is an improved version of the RFP coding device BBa_J04450. In this composite part, the expression of the mRFP1 protein is under the control of a Lac promoter (BBa_R0010), which is repressed by LacI and induced by IPTG. Furthermore, the composite part consists of a ribosome binding site (RBS) (BBa_B0034) and a double terminator (BBa_B0015). The RBS is an upstream (5’) component of a gene fragment and functions to bind the ribosome efficiently, to subsequently generate a higher control and accuracy over mRNA translation initiation. For efficient protein production in bacteria, most genes require an RBS and a start codon [3,4]. The RBS is located in the gene between the LacI promoter and the gene fragment coding for mRFP1. By optimization of the RBS region between the LacI promoter and the mRFP1, a higher level of protein expression can be obtained.

Translation enhancing 5’UTR (BBa_K1758100)

To obtain higher protein expression, a translation-enhancing gene component was designed by the iGEM team of Bielefeld-CeBiTec 2015, additionally to the ribosome binding site. Part BBa_K1758100 is such a sequence, containing a 5’ untranslated region (5’UTR). This part is already proven to increase the expression of the consecutive gene, compared to the standard RBS BBa_B0034 [5]. Furthermore, the research paper of Volkenborn et al. [6] confirmed the importance of a 5’UTR sequence for translation initiation. Part BBa_K1758100 contains different components, which together increase protein expression. A poly-A-spacer is added, which recruits the ribosome by recognition of the 5’UTR mRNA [7]. In front of the poly-A-spacer a T7 gene 10 leader (g10-L) sequence is present, which enhances the expression of foreign genes in E. coli cells [8]. To improve the RBS of the RFP coding device (BBa_K801100), the previously mentioned translation enhancing components are combined in part BBa_K3972006. This part is designed to optimally enhance protein expression in prokaryotes and it consists of translation enhancing DNA, a 10-A-spacer, a g10-L RBS and an AT-rich region, as shown in Figure 1.

Figure 1: Overview 5’UTR gene sequence.

Comparing RFP coding device with RBS (BBa_K801100) & RFP coding device with translation enhancing 5’UTR (BBa_K3972005)

We combined the above-mentioned part BBa_K3972006 with the sequence of the RFP coding device BBa_K801100, to design a new DNA sequence (BBa_K3972005), which enhances the expression of mRFP1. The BBa_K801100 sequence is improved by the replacement of the RBS (BBa_B0034) by a translation enhancing 5’UTR with a g10-L RBS. The gene segment contains a 10-A-spacer, followed by a ribosome binding site, to increase expression of mRFP1 (BBa_E1010).

Part BBa_K3972005 was ordered from IDT with additional RFC[10] BioBrick compatible restriction sites and was successfully ligated in a pSB1C3 plasmid, provided by iGEM in the 384-well plates. To characterize the E. coli optimized RFP coding device containing a 5’UTR (pSB1C3-5’UTR-RFP), different experiments were executed. Due to leaky expression of RFP in the original RFP coding device (BBa_J04450), we expected the bacterial colonies to be pink.

As can be seen in Figure 2a, the pSB1C3-5’UTR-RFP plasmid was successfully transformed into E. coli TOP10 cells. Three different colors of colonies were observed, shown in the red, pink and light blue circle. Furthermore, a difference in fluorescence intensity was observed for the same colonies under UV light (Figure 2b). From colonies present on the negative control, restricted pSB1C3 plasmid (Figure 2c), it can be concluded that not all plasmids were properly restricted. The light pink and white colonies present on the plate with the pSB1C3-5’UTR-RFP plasmid are probably unrestricted pSB1C3 plasmid. The brightest colonies were used to grow small cultures.

The small culture of the original pSB1C3-RFP plasmid (BBa_K801100) and of the pSB1C3-5’UTR-RFP plasmid (BBa_K3972005) can be seen in Figure 3. These small cultures were grown starting at the same time, for approximately 22 hours and were not induced. A proper color difference can be observed between the two, which already indicates faster mRFP1 expression by the bacteria containing the pSB1C3-5’UTR-RFP plasmid. More details can be found on the Part Registry pages.

Figure 2: a) E. coli TOP10 cells with pSB1C3-RFP plasmid, b) E. coli TOP10 cells with pSB1C3-5’UTR-RFP plasmid under UV light, c) E. coli TOP10 cells with restricted pSB1C3-RFP plasmid (negative control).

Figure 3: Small cultures wild type pSB1C3-RFP plasmid (right) and improved pSB1C3-5UTR-RFP (left) in E. coli TOP10 cells.

In conclusion, looking at the grown colonies on the agar plates, the grown small cultures and literature, it can carefully be claimed that faster mRFP1 expression occurs when the RBS (BBa_B0034) is replaced by an 5’UTR RBS (BBa_K3972006). However, further fluorescence intensity measurements have to be executed.

The functionality of the 5’UTR RBS gene fragment to generate higher protein expression, can be applied to our designed sensor-reporter system, described in Proof of Concept segment Sensor, to enhance the gas vesicle concentration. The DNA sequence of design B contains the RBS BBa_B0034, in front of the ARG1 sequence. By removing the original RBS and replacing it by our designed 5’UTR RBS BBa_K3972006 the expression of the ARG1 gas vesicle proteins could be increased. This will be an advantage for our final application IBDetection, since higher protein expression will lead to more and faster expressed gas vesicles. This will lead to better ultrasound images and a shorter time before gas vesicle measurements can be applied. For details see Project Description segment Our Solution

Part Table

Part Number	Type	Name	Description	Length	Favorite
BBa_K3972005	Composite	5’UTR-RFP	This part is an improved part of the RFP Coding Device BBa_K801100, which is improved by the replacement of the RBS BBa_B0034 by a translation enhancing 5’UTR containing a g10-L RBS BBa_K3972006.	1089 bp	❤
BBa_K3972006	Basic	5’UTR with g10 RBS	This part is used to improve the protein expression of part BBa_K801100. The part consists of translation-enhancing DNA, a poly-A-spacer, an RBS, and an AT-rich region.	54 bp

References

Müller-Taubenberger A, Anderson KI. Recent advances using green and red fluorescent protein variants. Appl Microbiol Biotechnol. 2007;77(1):1–12.
Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, et al. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A. 2002;99(12):7877–82.
Ribosome Binding Site/Cat page [Internet]. Igem.org. [cited 2021 Oct 17]. Available from: http://parts.igem.org/Ribosome_Binding_Site/Cat_page
Omotajo D, Tate T, Cho H, Choudhary M. Distribution and diversity of ribosome binding sites in prokaryotic genomes. BMC Genomics. 2015;16(1):604.
Takahashi S, Furusawa H, Ueda T, Okahata Y. Translation enhancer improves the ribosome liberation from translation initiation. J Am Chem Soc. 2013;135(35):13096–106.
Volkenborn K, Kuschmierz L, Benz N, Lenz P, Knapp A, Jaeger K-E. The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis. Microb Cell Fact. 2020;19(1):154.
Hellen CU, Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 2001;15(13):1593–612.
Olins PO, Devine CS, Rangwala SH, Kavka KS. The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Gene. 1988;73(1):227–35.

Team:TU-Eindhoven/Improve