Part
Improvement

"There's always room for improvement no matter what" - Ali Krieger

Introduction

5' UTR-BA_K1758100

BBa_I746909

BBa_K4160016 

Aim

Methods

Results

Discussion

References


The iGEM team Eindhoven 2022 improved the part BBa_I746909 by removing the RBS and adding a 5’ UTR( BBa_K1758100) in its place during the design phase. The 5’ UTR created by iGEM 2015 team Bielefeld contains a 5' UTR and a strong RBS (ribosomal binding site) from bacteriophage T7, which is called g10-L. Both the UTR sequence and the RBS sequence enhance the translation of the desired protein. 2-4


Below an introduction of the parts can be found followed by a caracterization.

Part Number Type Name Description Length
BBa_I746909 Basic sfGFP Folding of Superfolder GFP is enhanced relative to the folding of wild-tyoe GFP 720bp
BBa_K1758100 Basic 5’UTR with g10-RBS This part is used to improve the protein expression of part BBa_I746909. The part consists of translation-enhancing DNA, a poly-A-spacer, an RBS, and an AT-rich region. 54bp
BBa_K4160018 Basic sfGFP with 5'UTR This part was developed for part improvement. The RBS in BBa_I746909 was replaced by BBa_K3972006 was added to. 54bp

5' UTR - BBa_K1758100

5' UTR (untranslated region) is a small piece of mRNA placed on the 5' end of the mRNA. When mRNA is translated into protein, the ribosome can bind at the 5’ site. The 5' UTR sequence directs effective translation and enhanced expression of the following gene. 1


The iGEM 2015 team from Bielefield created such an UTR. This part contains a 5' UTR and a strong RBS (ribosomal binding site) from bacteriophage T7, which is called g10-L. Both the UTR sequence and the RBS sequence enhance the translation of the desired sequence.
More specifically, the part contains a T7 g10 leader sequence, which enhances the efficiency of translation in E. coli cells. Secondly, a poly-A-spacer enhances the recognition of the mRNA sequence by the ribosome. Thirdly, The RBS is followed by an AT rich region, which continues until the start codon, to enhance resulting protein translation. 2-4


Figure 1 | sequence of 5' UTR developed by 2015 iGEM team Bielefield. The pink part it the T7 leader sequence. The light green part is the poly-A-spacer. The dark green part is the RBS. the yellow part it the AT rich region.

Superfolder GFP driven by T7 promoter - BBa_I746909

Fluorescent proteins have a lot of biological applications. For example, it can function as a reporter when coupled to a protein of interest. With this application, protein distribution inside the cell can be visualized. Green fluorescent protein (GFP) is such a fluorescent protein. An example of a GFP is the part superfolder GFP, driven by T7 promoter, and this part is Biobrick compatible. sfGFP is a protein that folds correctly, even under poor circumstances. This stable folding is the result of the mutations F64L and S65T, which are called enhanced GFP mutations. Besides, sfGFP contains the cycle 3 mutations which result in higher expression. This part also contains a T7 promoter, a ribosomal binding site, T7e, and a terminator (Figure 2). sfGFP is brighter than regular GFP and therefore it is more useful in practical use. 5,6


Figure 2 | BBa_I746909. This part consists of a T7 promoter, RBS, sfGFP and T7e with terminator

Superfolder GFP containing 5’UTR - BBa_K4160016 

Combining the parts mentioned above will create part BBa_K4160016. This part contains the 5’ UTR (BBa_K1578100) followed by sfGFP(BBa_I746909). We hypothesize That combining these parts together, will further enhance expression of sfGFP, resulting in a higher fluorescent signal. Enhancing the fluorescence of fluorescent proteins can be useful to localize enzymatic an regulatory processes. 7


Figure 3 | BBa_onze part. Part containing T7 promoter, UTR, sfGFP and T7e with terminator

Aim

The goal was to develop a part for the high school challenge day. This is an educational event organized by our team for high school students to interact with synthetic biology. During the challenge day high school students went into the lab and created drawings with fluorescent bacteria, among which was sfGFP. We planned to enhance the expression level in order to obtain a stronger fluorescent signal which would create better visible drawings.

Methods

Figure 1 | Cloning strategy for Part improvement. . The RBS sequence is restricted from part BBa_I746909, after which the sequence encoding for UTR (BBa_K1758100) is ligated. This will result in our new part (BBa_ K4160018), with expected higher expression of sfGFP.

The first step was choosing a plasmid. For this experiment, the plasmid pSB1C3 from the distribution kit 2022 plate 1 in well 1O was chosen. To increase the amount of plasmid, the DNA was transformed in E. coli DH5α cells and the amplified plasmid was isolated

Both parts, sfGFP( BBa_I746909) and sfGFP + UTR( BBa_K4160018), were retrieved from IDT. Via restriction and ligation the parts were implemented into the Psb1C3 plasmid. Restriction enzymes EcoR1-HF(NEB) and Spe1-HF(NEB) were used. The design of sfGFP+UTR can be seen in figure 1.

Both plasmids, sfGFP and sfGFP with UTR, were transformed into E. coli BL21 cells (Thermofischer). These BL21 cells are specialized for protein expression. Figure 2 and 3 show the LB-agar plates containing grown bacterial cultures of the transformed BL21 cells. After the overnight incubation, small cultures were started from the cultures grown on the LB-agar plate. The next day large cultures were started.

Both plasmids, sfGFP and sfGFP with UTR, were transformed into E. coli BL21 cells (Thermofischer). These BL21 cells are specialized for protein expression. Figure 2 and 3 show the LB-agar plates containing grown bacterial cultures of the transformed BL21 cells. After the overnight incubation, small cultures were started from the cultures grown on the LB-agar plate. The next day large cultures were started.

Figure 2 | LB-Agar plate with sfGFP and UTR after transfection
Figure 3 | LB-Agar plate with sfGFP after transfection

The large culture was started by taking a 2500 mL culture flasks, adding 250 mL 2YT, and the 8 mL small culture. OD600 was measured after one hour, which already exceeded a value of 0.6. Next, expression was induced by adding 250 µL IPTG and 250 µl chloramphenicol antibiotic. After induction, 1 mL samples were taken every 20 minutes, and snapfreezed in liquid nitrogen for storage in the -80° C freezer. After 5 hours of sample collection, we thawed all of the samples and measured the fluorescence using the platereader, at emission wavelength 510 nm. The making of the large culture was done in duplo and both results can be found in the results section.

Results

Figure 4 | Large culture samples. sfGFP and sfGFP + UTR after 5 hours of expression shown under blue light. The Eppendorf tube on the left contains sfGFP with UTR and the Eppendorf tube on the right contains sfGFP. Samples were exited using a 470 nm blue light illuminator.




Figure 5 | fluorescense of sfGFP with UTR and sfGFP over time. The sfGFP is colored green and the sfGFP with UTR is colored blue. Measured by a platereader at emission wavelength 510 nm, using technical triplets.

Figure 4 and 5 show the results from the first experiment. Figure 4 shows two 1mL samples taken from each large culture after 5 hours. Samples were excited using a 470 nm blue light illuminator. Figure 5 shows the fluorescence of all 1 mL samples taken from each large culture within a 5 hour timespan. Fluorescence was measured using the plate reader at emission wavelength of 510 nm. The green line resembles sfGFP and the blue line resembles sfGFP with UTR. This graph was made using technical triplets.

Figure 6 | sfGFP and sfGFP + UTR after 5 hours of expression shown under blue light. The Eppendorf tube on the left contains sfGFP with UTR and the Eppendorf tube on the right contains sfGFP. Samples were exited using a 470 nm blue light illuminator.
Figure 7 | fluorescense of sfGFP with UTR and sfGFP over time. The sfGFP is colored green and the sfGFP with UTR is colored blue. Measured by a platereader at emission wavelength 510 nm, using biological triplets.

The experiment was repeated in duplo because previous results were unexpected. The results from this experiment can be found in figure 6 and 7. In figure 6, on the left the Eppendorf tubes are shown containing sfGFP and on the right the Eppendorf tubes containing sfGFP with UTR. The graph in figure 7 shows measured fluorescent signal, in a timeframe of 5 hours. This time biological triplets are taken.

Discussion

The experiments were performed in duplo because during the first experiment unexpected results were obtained, probably due to the OD600 of the sfGFP. A higher OD600 means a higher concentration of cells, resulting in more protein expression leading to a higher fluorescence The OD600 was measured 0.9 for sfGFP compared to the OD600 from sfGFP with UTR from 0.6. When redoing the large culture, the starting values for OD600 were 0.6 for both sfGFP and sfGFP with UTR.


As can be seen in figure 4,5,6 and 7, a yellow less fluorescent color can be observed. We did not expect a weaker fluorescence, as previous parts where the UTR was added showed a clear enhancement of expression (For example, ( BBa_K3187014). Nevertheless, the sfGFP with UTR showing a more yellow color and lower fluorescent signal, could be useful for future drawing projects, since the color pallet could be expanded with this sfGFP variant.


Using commercial sanger sequencing (baseclear) the sequence used for the experiment was checked for presence of the UTR sequence in the UTR sample. Furthermore, the sfGFP gene was checked if mutations were present, leading to formation of YFP. However, no mutations were found.

An important aspect of this part, is that it should be ligated into plasmid pSB1C3. In figure 3 can be seen that this plasmid is not pSB1C3. This plasmid was retrieved from the distribution kit, plate 1, well 1O. Unfortunately it was not pSB1C3 but a different plasmid, we noticed that this plasmid was not present in the well and we obtained some other plasmid.
This could be concluded after a 1x digestion, then a linear plasmid is created. Since the Psb1c3 is around 2000 pb a band around 2.0 kb is expected. Unexpectedly, a band around 3000 bp is seen this means this is not the expected plasmid.

Outliers in figure 7 could be explained by interchanging different samples, leading to unreliable results. One tube from time 160 and 200 have been switched.

Figure 8 | Agarose gels analyses after DNA separation of digested plasmids and inserts. After digestion of pSB1C3 (EcoR1-HF and Spe1-HF, 2050 bp), GFP and UTR + GFP(EcoR1-HF and Spe1-HF, respectively, 923 bp and 963 bp). An analyses on the plasmid pSB1C3. It is ran on an agarose gel in a non digested, 1x digested and 2x digested form. The linear(1x digestion) plasmid shows a clear band at 3000 bp, meaning this plasmid is not the expected plasmid(pSB1C3) The outlined DNA bands were cut out and purified. Agarose gel ran for 1 hour at 100V in 1x TAE buffer, stained with SYBR Safe DNA gel stain. Gel analyses was performed with a 470 nm blue light illuminator.
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