Reporter

Part:BBa_K3944007

Designed by: Olof Dahlman   Group: iGEM21_Chalmers-Gothenburg   (2021-09-20)


ScCUP1 + miRFP670
Copper promoter CUP1 followed by red fluorescent protein miRF670, which acts as a reporter for the level of induction. The sequence has been codon optimized for use in S. cerevisiae and requires the native yeast protein ACE1 to function. optimised for use in S.Cerevisiae and requires the native yeast protein ACE1 to function.

Sequence and Features


Assembly Compatibility:
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    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 467
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 467
  • 21
    COMPATIBLE WITH RFC[21]
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    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 467
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    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 467
  • 1000
    COMPATIBLE WITH RFC[1000]

Aims

Our team aimed to construct a yeast strain where the fatty acids production profiles could be tuned by the use of different chemical induction systems. Our proposed method involves three different induction systems linked to different thioesterases. Thioesterases are proteins that terminate fatty acid synthesis by hydrolyzing acyl-CoA and have preference for different chain lengths. In order to verify the function of our system we tested the induction systems by expressing fluorescent proteins.

In addition to this copper-dependent induction plasmid expressing miRFP670 with red fluorescence, we also constructed and tested the induction systems TetOn with BFPand Estradiol with GFP.

Usage and Biology

The copper-induced promoter CUP1 is native to yeast and is activated when the copper-binding protein ACE1 (2) binds to it following ligand binding to copper. The yeast then expresses metallothioneins which bind and sequester potentially harmful metall ions, such as CU2+. A small amount of copper is necessary to the growth and survival of the yeast however, and as such most growth media contain it and this system tends to have leaky expression.

Since the system is native to yeast, any gene of interest need only be inserted behind the CUP1 promoter and transformed or integrated into the yeast to build a functional, if leaky, inducible expression system. Thioesterase induction the native yeast FAS system needs to be replaced prior however, as the native FAS1 system is a dimer of two large protein strands forming a large complex, which includes thioesterase, leading to competition between native and induced thioesterase without any ability for detailed control.

Experimental design

Copying the CUP1 promoter and miRFP670 sequences from template via PCR allows for the insertion of overlapping overhangs at the primer design stage, which enables us to make use of quick and efficient one-pot Gibson assembly reaction to insert the promoter and thioesterase in the plasmid backbone p413TEF. 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.

Methodology

Templates of CUP1 and TesBT were ordered from IDT and mixed with the corresponding forward- and reverse primer pairs. PCR reaction performed with Phusion polymerase according to protocol. 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 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, inoculated and then harvested with miniprep. The harvested plasmids were sequenced and transformed into yeast resulting in seven different strains described as 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. Due to inadequate ability to detect blue fluorescence with this machine all cells containing TetON-BFP had to be measured using a Sony FACS machine. The FACS machine is a manual machine where each sample is analyzed separately, limiting the amount of induction cell/inducer combinations and experiments that could be performed for all strains with TetON-BFP. Thus, data availability is greater for samples with either copper, estradiol or both. During the experiments, the instrument was set up to compensate for overlaps in excitation/emission spectra between the fluorophores (particularly the problematic BFP/GFP emission/excitation overlap). The results were analyzed to determine expression efficiency.

Results

Single plasmid experiments were conducted, where fluorescence from each individual plasmid was tested with different inducer concentrations.


Single Copper plasmid

RFP expression is dependent on the copper inducer concentration. Figure 1 displays violin plots of the logarithmic values for red fluorescent expression when adding the copper induction agent in concentrations of 1, 10, 50, 100 µM for experiment A and 0, 100, 500 and 1000 µM for experiment B. The right side of figure1 provides control values when different concentrations of inducers are added to wild-type yeast.


There is a clear indication that our induction plasmid provides more red fluorescence than the native yeast (control), meaning that the system would provide an output. However, it is clear that the copper induction system is leaky due to the high expression that both experiment A and B shows for the test without any induction agent, located to the far left. Ideally, there would be no signal from the uninduced sample above the signal from the negative control. Still, there is an increase in expression for the higher concentrations of inducer, the difference being especially clear in experiment B for the 500 µM case.


Chalmers Gothenburg 2021 Violin cup RFP ex2.png

Figure 1: Violin plots of RFP expression for single plasmid in yeast strain CENPK102-5B, from two experiments, A and B. Measured with Guava. 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)


To further clarify the differences in fluorescent expression, the RFP intensity was plotted against density of measurement points for different doses in figure 2. These density plots provide a similar result to figure 1 with very similar RFP intensity. However, the density and intensity provided in the two bottom graphs displays more clearly that the concentration 100 µM for experiment A and 500 µM for experiment B has a higher expression. The key aspect is shown by comparing the position of the curve peak, where the more right it is, the stronger the expression.


Chalmers Gothenburg 2021 Density Lincoln cup RPF two.png


Figure 2: Overlapping density plots of RFP expression for all concentrations of copper inducer. Measured with Guava.


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 Copper and Estradiol induction

The results from the yeast strain with the copper and estradiol dual plasmid system are displayed in figure 3. The left violin plot shows the GFP expression for different estradiol inducer concentrations (Est) and the right plot shows the RFP expression for different copper inducer concentrations (Cup). The violin plot for GFP shows clearly that the expression increases when estradiol is added, with or without the copper inducer. There also does not seem to be a change in expression between the samples with 0/0.01 µM (0 µM copper inducer and 0.01 µM estradiol inducer) and 100/0.01 µM, indicating that the system is not affected by the copper. The right plot for the RFP expression is, however, not as clear as the estradiol one. The RFP expression does not seem to change much for either of the inducers, but there is a slight increase when comparing the 100/0.01 sample with the 0/0 sample, but it is not as distinct as in the estradiol case.

Chalmers Gothenburg 2021 violin cup.est GFP RFP2.png

Figure 3: Violin plots of GFP (GRN.B.HLog) and RFP (RED.R.HLog) from yeast strain containing copper and estradiol plasmids. Measured with Guava. 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)


For additional clarifications there are also density plots for green and red fluorescent expression provided in figure 4. Both of the plots on the left show the GFP expression, and it is rather clear in both graphs that the expression increased as the estradiol inducer is added. The bottom one also shows that the expression does not change as the copper inducer is added, just as the violin plots also did. The RFP on the right side of figure 4, is on the other side of the spectra, and is not as clear. However, the bottom graph displays more clearly that the RFP expression is higher for the sample with both copper and estradiol, which indicates that the copper system increases in expression when estradiol is added.


Chalmers Gothenburg 2021 Density Lincoln cup.est.png

Figure 4: Overlapping density plots for BFP and RFP expressions from yeast strain containing both copper and estradiol plasmids. Measured with Guava.

A comparison of the given fluorescent expression for GFP compared to controls is given in figure 5. The graph shows a large difference in expression between native yeast and transformed one, corroborating the notion that the system leaks. The most interesting aspect is, however, that there is no difference in expression between case and control with 0.01 µM estradiol and/or 100 µM of copper, meaning that the estradiol system is not affected by the copper inducer.

Chalmers Gothenburg 2021 violin cup.est GFP2.png

Figure 5: Violin plots of GFP expression from three yeast strains, one case and two controls. Measured with Guava. 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)


A similar violin plot for RFP with controls, is displayed in figure 6. The result from this figure shows a great difference in expression if a plasmid is integrated (case) or not (CENPK control) into the yeast, showing the leakage of the system. The most important aspect in this figure is the difference in expression between the 100/0.01µM samples. For the control with only the copper plasmid integrated, the expression is much lower than for the yeast with both estradiol and copper plasmids, providing additional results that support the notion that the copper system is affected by the presence of the estradiol plasmid


Chalmers Gothenburg 2021 violin cup.est RFP2.png

Figure 6: Violin plot of RFP expression from three yeast strains, one case and two controls. Measured with Guava. 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)


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 7. 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.

Chalmers Gothenburg 2021 violin Tet.cup BFP RFP2.png

Figure 7: 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 7 are given in figure 8. 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.


Chalmers Gothenburg 2021 Density Lincoln tet.cup.png

Figure 8: 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 9. 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.


Chalmers Gothenburg 2021 violin Tet.cup.est2.png

Figure 9: 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 9 is shown in figure 10, where the close expression overlap shown in the violin plots from figure 9 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.

T--Chalmers-Gothenburg--Density_Lincoln_tet.cup.est.png

Figure 10: 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.

Discussion

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.



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