Benchmarking device used to measure induced transcription by tetracycline induction system using blue fluorescent protein as a reporter. This system requires the presence of the tetracycline-sensing protein TetA to function, see here. For an overview of the whole system, please visit our wiki.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI site found at 5
We set out to construct a yeast strain that could be altered or programmed on the fly to produce different types of fatty acid. Our proposed method involves three different induction systems linked to different thioesterases, which are proteins that terminate fatty acid synthesis at specific lengths by cleaving off the ACP moiety from the growing chain, determining its length dependent on the preference of the enzyme. In order to verify the function of our system, we constructed induction systems expressing fluorescent proteins, making for easy verification via fluorescence measurements. In addition to this tetracycline-dependent induction plasmid expressing mTagBFP2 with blue fluorescence, we also constructed and tested induction systems Copper with RFP and Estradiol with GFP.
Usage and Biology
The blue fluorescent protein mTagBFP2 emits a blue colour upon being expressed, and is useful as a report of successful expression (1). It is derived from Entacmaea quadricolor and has been shown to have high chemical stability (2).
The tetracycline transcriptional system originates from bacterial antibiotic resistance genes, reacting to the addition of members from the tetracycline family of antibiotics. In bacterial origins, it triggers transcription of genes downstream of TetO sequence repeats + a promoter of choice, conferring antibiotic resistance to the cell. By moving this system to other organisms, a reliable if somewhat leaky way of expressing desired genes can be obtained. Seven TetO repeats has been found to be optimal (3) and by placing these before a promoter of choice, such as ScPHO5, the downstream gene will be transcribed. As the system requires the introduction of the tetracycline-sensing protein, building a single plasmid with both the promoter/gene system and constitutively expressed sensing protein, a single-transform induction system can be created, making use much easier.
The seven TetO repeats were obtained on a plasmid with the ScPHO5, optimized for use in S.Cerevisia. By designing primer pairs that leaves the TetO/ScPHO5 and mTagBFP2 sequence with overlaps to one another, the TetA sensing protein and the p415TEF plasmid backbone, the plasmid can be assembled in a one-pot Gibson assembly reaction, which has been shown to work with up to 12 insert fragments, and designed as such that the promoter/gene region is inserted in the reverse direction to avoid potential transcribing/overlap errors with the TetA sensing protein. The created plasmid is then ready to use in benchmarking experiments, where we prepared cells of S.Cerevisiae strain CEN.PK 102-5B with all possible plasmid combinations: Single cells with CUP1, TetON and Estra, Double cell lines with CUP1-TetON, CUP1-Estra and TetON-Estra and a final tripple strain with CUP1-TetON-Estra. Measuring the fluorescence of these cell strains not only allow us to compensate for overlaps in fluorescence spectra to obtain accurate signals, but also allow for the measurement of the interactions between induction systems and how the presence of several systems in a yeast cell affect its viability.
Templates of TetO7/ScPHO5 and mTagBFP2 ordered from IDT or provided by our department were mixed with the corresponding forward/reverse primer pair and PCR reaction performed with Phusion polymerase according to protocol to create the inserts. Backbone-containing Backbone-containing E.coli cells were grown overnight and then harvested by following protocol for ThermoFischer plasmid miniprep kit, and subsequently cleaved with restriction enzyme to linearize the backbone. The created insert fragments and cleaved backbone were purified using gel purification and ThermoFischer gel purification kit, followed by assembly using the Gibson assembly method according to protocol. The assembled plasmid was transformed into competent E.coli of strain DH5-alpha, grown overnight, inoculated to be grown overnight again and then harvested with miniprep. The harvested plasmids were sequenced and then transformed into the yeast cells to create the seven different strains described: CUP1, TetOn, Estra, CUP1-TetON, CUP1-Estra, TetON-Estra and CUP1-TetON-Estra. CUP1 and Estra-containing strains were measured using GUAVA flow cytometer machine, but due to inadequate ability to detect blue fluorescence, all cells containing TetON with BFP had to be measured using a Sony FACS machine. The FACS machine is a manual machine where each sample is analysed separately, limiting the amount of induction cell/inducer combinations and experiments that could be performed for all strains with tetracycline/BFP plasmid. Thus, data availability is greater for samples with either copper, estradiol or both. During the experiments, the instrument was setup as to compensate for overlaps in excitation/emission spectra between the fluoropores, particularly the problematic BFP/GFP emission/excitation overlap, eliminating signal distortions from this source. The results were collected as excel files and processed to determine expression efficiency.
Single plasmid experiments were conducted, where fluorescence from each individual plasmid was tested with different inducer concentration
Single Tetracycline plasmid
Our tetracycline induction system is connected to the expression of BFP. The measured expression from the single plasmid analysis is given in figure 1 as violin plots. The data shown in this graph point to a great difference in intensity compared to the negative control, making it rather clear that the tetracycline system is leaking, given the high expression with the inducer concentration at 0 µm. It can still be seen that there is a slight increase in expression of BFP when the inducer concentration increases.
Figure 1: Violin plot of BFP expression from single induction plasmid in yeast strain (CENPK 102-5B). Measured with FACS. Pairwise statistical tests between controls (0uM added inducer) and remaining samples in each strain were performed using unpaired two-sided Wilcoxon-rank-sum tests (ns: not significant, *p < 0.05, **p<0.01,***p<0.001,****p<0.0001)
The results from figure 1 show that the induction system is to some extent concentration-dependent, illustrated more clearly with the density plots in figure 2. Both the top and bottom graphs show a slight increase in expression when the concentrations of inducer increase. It is more visible in the bottom graph that, without a complete overlap, the higher concentration of tetracycline, the more to the right the curve is shifted.
Figure 2: Overlapping density plots of BFP expression for all concentrations of estradiol inducer. Measured with Guava.
Conclusion single plasmid
All three induction systems show an inducer concentration-dependent behaviour. Both the copper system and the tetracycline system show very high leakage levels, but they also show an increase in expression with higher concentrations of inducer. The estradiol system also shows indications of leakage, but compared to the actual output signal when inducers are added, the leakage is relatively small.
Combinatorial benchmarking (two induction systems)
Although the single plasmid results indicate that the induction systems are functional, it does not explore the potential effect they could have on each other. For this reason, we also tested dual plasmid combinations in the same fluorescence manner as the single plasmid systems. The GFP and RFP combination was tested using the Guava flow cytometer while the others with BFP were tested using the FACS machine.
Dual plasmids of Tetracycline and estradiol induction
The results from the yeast strain containing both tetracycline and estradiol plasmids are given in figure 3. The left violin plot shows the logarithmic value of TetON-BFP (log_BFP), for different tetracycline inducer concentrations, while the left shows the Estra-GFP expression (log_EGFP), for different estradiol inducer concentrations. The provided BFP results shows, as discussed for the single plasmid results, that the tetracycline system is leaking due to the similar expression both with and without inducer. The GFP expression for estradiol is also rather similar for non-induced sample and induced sample, though the expression is higher in intensity for the induced sample.
Figure 3: Violin plot of BFP and GFP from yeast strain with tetracycline and estradiol plasmids. Measured with FACS.
The density plots of the same samples are provided in figure 4, where the expression for both BFP and GFP is clearer than in figure 3. The bottom plot on the left for BFP shows a higher expression when the system is induced then when it is not. Thus, the inducers will change the expression slightly when added. For the estradiol system the highest GFP expressions are only shown for the induced sample, indicating a change when inducers are added. There is still a large portion of the expression overlapping between the samples, showing leakage.
Figure 4: Overlapping density plots for BFP and GFP expressions from yeast strain containing both copper and estradiol plasmids. Measured with FACS.
Dual plasmids of Tetracycline and copper induction
The results given for the dual plasmids of tetracycline and copper are displayed in the violin plot in figure 5. The left plot shows the TetON-BFP expression (log_BFP), where there is a slightly higher expression for the induced sample than the non-induced one. Similar results are displayed in the right plot for Copper-RFP (log_miRFP670), where the expression is higher for the induced sample than the non-induced. The differences in expression between induced and non-induced are not large.
Figure 5: Violin plot of BFP and GFP from yeast strain with tetracycline and estradiol plasmids. Measured with FACS. Pairwise statistical tests between controls (0uM added inducer) and remaining samples in each strain were performed using unpaired two-sided Wilcoxon-rank-sum tests (ns: not significant, *p < 0.05, **p<0.01,***p<0.001,****p<0.0001)
Density plots for BFP and RFP from the same samples as in figure 5 are given in figure 6. In both cases of BFP and RFP the expression from induced and non-induced samples is overlapping, but there is a slight difference where the induced one is shifted slightly more to the right. This is especially clear for the bottom graphs, indicating a slightly higher expression for the induced samples.
Figure 6: Overlapping density plots for BFP and RFP expressions from yeast strain containing both copper and estradiol plasmids. Measured with FACS.
Conclusion dual plasmid measurements
The results from the dual plasmids displays a somewhat clear increase in fluorescent protein expression in an induced sample compared to a non-induced one, indicating that the system is functional to some degree. There still is, however, a noticeable expression from the non-induced sample for more or less all strains, showing that all three systems leak to some degree. Additionally, the estradiol was not affected by the presence of the copper plasmid, as it provided the same signal with or without copper inducer, while the opposite could be said for copper, where the overall output signal was higher for strains with the estradiol plasmid even when there was no copper inducer present.
Overall, it was observed that the system works for dual plasmids reasonably well despite leakage, providing data that the plasmids and systems work together.
Combinatorial benchmarking (three induction systems)
Fluorescent expression analysed for the strain containing all three induction plasmids are given as violin plots in figure 7. The plots show the three fluorescent expressions separately for three different inducer concentrations. The TetON-BFP (log_BFP) plots shows an increase in expression when the tetracycline inducer is added compared to when it is not added, though the difference between the two concentrations of inducers does not seem to affect the system greatly, but the increase in expression with the added inducer shows that the system is still functional to some degree with all three plasmids. The plot in the centre for Estra-GFP (log_EGFP) expression does not change between the non-induced sample and the induced ones, indicating that either the system is inhibited by the other plasmids or that the plasmid has dropped out of the cell. For the right and last plot, the expression of Copper-RFP (log_miRFP670) is displayed. The expression increases as the copper inducer is added, indicating that the system is still functional. As previously shown, the RFP induction did increase as the estradiol inducer was added, but this is not the case in this experiment, which means that the copper system might only be affected by the other plasmids rather than other inducers.
Figure 7: Violin plot of BFP, GFP and RFP from yeast strain with tetracycline, estradiol and copper plasmids. Measured with FACS. Pairwise statistical tests between controls (0uM added inducer) and remaining samples in each strain were performed using unpaired two-sided Wilcoxon-rank-sum tests (ns: not significant, *p < 0.05, **p<0.01,***p<0.001,****p<0.0001)
Density plots of the same data as in figure 7 is shown in figure 8, where the close expression overlap shown in the violin plots from figure 7 becomes extra clear. The slight increase in expression for both BFP and RFP is especially clear in the bottom graphs, where the induced samples are located more to the right or higher up on the expression axis. The GFP density plot in the middle is almost identical for all samples.
Figure 8: Overlapping density plots for BFP,GFP and RFP expressions from yeast strain containing both Tetracycline, estradiol and copper plasmids. Measured with FACS.
Conclusions combinatorial benchmarking
The overall results from the triple plasmid system shows a slight increase for TetON-BFP and Copper-RFP when inducers are added, but not for the Estra-GFP, meaning that the system works for BFP and RFP, but not for GFP. The results from the dual and single plasmid systems shows that the estradiol system functions the best out of the three systems, and it should translate to the triple plasmid system. The single plasmid systems also showed that the estradiol plasmids most likely has a faster plasmid drop-out rate, which could be the reason for the estradiol system results.
The combinations of all experimental data for the plasmid induction systems shows that all three systems should work rather well, with some leakage, and also shows that the plasmids are affected when more plasmids are present in the same cell. Interestingly enough the results provided by the guava machine showed a much clearer expression separation for different inducers than the FACS, which is something that could be taken into consideration when additional testing is performed in the future. Another beneficial part of using the guava is the automatic sampling from a microplate, which helps increase the amount of different inducer concentrations when needed. Therefore, for a future perspective, when benchmarking a precise amount of inducer for a specific output signal a wellplate reader such as the guava will be the better choice and the BFP gene should be replaced to ensure compatibility.
Another important aspect that needs to be tested in the future, is in which growth phase each inducer is the most efficient and has the best function. Because as of now each inducer were tested using the same procedure, expect for experiment A for single plasmids of copper and estradiol which suffered from some time lag, further described in the notebook, though essentially, the cells were cultivated for a longer period of time before being measured in the guava, which might be a reason for the higher numeric value of expression of experiment A compared to experiment B in figure 1 and 3.
To summarize the results, all systems were proven to work to some degree, and to be dependent on concentration of added induction agent, which are the key results for the future of our project.