Difference between revisions of "Part:BBa K4160018"

Revision as of 15:01, 11 October 2022

sfGFP with 5-UTR

This is an improved part of the superfolder GFP driven by T7 promoter ( BBa_I746909). This is done by removing the RBS and adding a 5’ UTR ( BBa_K1758100) in its place.

Usage and biology

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 protein. An example of a GFP is the part superfolder GFP driven by T7 promoter, this part is Biobrick compatible. An sfGFP is a protein that folds correctly, even under poor circumstances. sfGFP is brighter than regular GFP therefore it is more useful in practical use.

Connecting sfGFP to a 5’ untranslated region(5’ UTR) will lead to even higher expression of the GFP. Therefore it is more visible and has more use. 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 sequence.

The combination the 5’ UTR with sfGFP is done via restriction and ligation. This can be seen in figure 1.
The initial goal with the part improvement was to enhance the expression of the sfGFP. With a side goal to use this part in a following challenge dayorganized by our team, this is an educational day for high school students. During the challenge day high school students went into the lab and created drawings with fluorescent bacteria, among which GFP. When enhancing the expression a more fluorescent color is received which would create better visible drawings.^1-3

Characterization

Methods

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 (Thermo Fisher Scientific). 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 (Thermo fisher Scientific). 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.

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 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.

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.

References

1. Lentini R, Forlin M, Martini L, et al. Fluorescent Proteins and in Vitro Genetic Organization for Cell-Free Synthetic Biology. ACS Synth Biol. 2013;2(9):482-489. doi:10.1021/sb400003y
2. Hellen CUT, Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 2001;15(13):1593-1612. doi:10.1101/gad.891101
3. Olins PO, Rangwala SH. A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989;264(29):16973-16976.