Team:ShanghaiTech/Project NFBL
Negative feedback loop
Introduction
Part B most importantly includes STAR. Among these components, STAR（Small Transcriptional-Activating RNA）is a significant switch which determines whether the gene of interest downstream could express or not. If STAR is absent, the structure inhibits expression of the gene downstream and there will be no transcription. On the other side, with the presence of STAR, it serves in trans and base pair to terminator RNA sequence, in this case, the function of terminator component is negated and downstream gene of interest will be transcribed. GFP is used to denote the status of STAR, giving a visual and distinct result.
Overview
Here we are expecting to use STAR as a significant part to realize the orthogonal system we design. There are obvious advantages for introducing STAR into our system. Firstly, its function can be realized with lower metabolic burden compared to the universal protein-based regulatory systems. Besides, the presence of STAR produces an expression of the downstream gene which is much higher than that when STAR is absent, which provides the efficiency and validity. Additionally, STAR acts quickly when receiving signal molecules and causes little delay in the circuit because of rapid RNA degradation.
Experiment design
The experiments on STAR can be generalized into two parts. Firstly, three different types of GFP are cultivated and examined using plate reader and fluorescence microscope, from this part of the experiment we verified the effectiveness of GFP in part B. Additionally, by comparing the light intensity of different types of GFP, part B is proved to be sensitive to embody its working status. The second part was designed to measure the growth and green fluorescence of cells under time span, in order to coordinate with data and curve from other parts in our experiment.
Result
Fig.1 The quantity of four different types of GFP (enhanced, control), which is shown to approach the same level.
Fig.2 The expression level of GFP decreases from enhanced group, STAR1 and STAR2 to control group, Blank(the fluorescence intensity are shown in logarithmic form).
Fig.3 The logarithmic form of fluorescence intensity divided by cell density respectively for STAR1, STAR2, Blank group.
By analyzing these two graphs, the result shows that the GFP used in part B is an enhanced GFP which intensity is nearly 10 times as the control group, which proves to be greatly sensitive. Meanwhile, this experiment proved part B works and creates a brighter image when observing under fluorescence microscope.
Fig.4 100x image of STAR1 under fluorescence microscope.
Fig.5 100x image of STAR1 under fluorescence microscope.
Fig.6 100x image of STAR1 under fluorescence microscope.
Plus, how STAR works according to time can be seen from the graphs below.
Fig.7 Bacterial density of STAR used in part B. The growth shows a steady increase from 0 to about 11h, then the numerical result fluctuates at approximately 1.04.
Fig.8 The expression quantity of STAR varies with time and stabilizes at about 15h (the red line is an estimated value, above which the fluorescence intensity of cell can be well detected.)
Fig.9 The ration of Fluorescence intensity divided by cell density. The curve first decreases sharply and then increases slowly, eventually stabilizes at about 15h.
Description
Quorum sensing is a natural occurring mechanism that certain strains of bacteria use to regulate gene expression in response to their population density. Signaling molecules such as N-acyl homoserine lactones or AHLs, could bind transcription factors and activate downstream gene expression. In our project, the Lux quorum sensing system was chosen to be the input signal controller of our Negative Feedback Loop (NFBL).
Design
The first node of our three-node negative feedback loop, Node A, was controlled by the Lux quorum sensing system and the repressive RNA switch pT181, which was designed as “pCon(J23100) + pT181 Target + RBS(BBa_B0034) + LuxR + TER(BBa_B0015)”.
We believe that, as pT181 RNA switch is open, the Lux transcriptional activator, LuxR, is translated. With the induction of exogenous CO-C6 AHL, LuxR will combine with AHL molecules and activate the Lux transcriptional promoter, pLux, and lead downstream gene expression.
Experiments
Experiments were carried out to determine which quorum sensing system should be chosen. We designed different plasmids which have different Quorum reporter devices and add green fluorescent protein at the downstream of Quorum transcriptional promoters to detect the performance of different quorum sensing concentrations.
Fig.10 pCon(J23100) + RBS(BBa_B0034) + QuorumR + TER(BBa_B0015) + pQuorum + RBS(BBa_B0034) + GFP + TER(BBa_B0015)
E. coli DH5α cells which contain plasmid of constructed reporter devices were cultured to Abs600=0.60 and then transferred to 96-well microplates where they were induced with appropriate AHL concentrations. The fluorescence output from each of the constructed devices was measured by inducing cell cultures with various concentrations of AHL molecules. The induced cell cultures were grown in the microplates at 37°C and the fluorescence signal were monitored over time by using a microplate reader.
We needed to characterize the response of the construct to different concentrations of AHL so that we could use the data in our model to predict how the system could function.
The activation ranges were compared between the quorum sensing systems in order to determine their robustness, sensitivity, and stability to see if it is suitable for use in our Negative Feedback Loop.
Results
Through experiments, concentration ranges of AHLs required for activation in each quorum sensing system were calculated to be 100nM-10uM for Rhl and 100pM-10nM for Lux, Tra, and Rpa. Rhl was found to differ by a 1,000-fold sensitive difference than other quorum sensing systems, which means Lux, Tra, and Rpa have higher sensitivity.
Fig.11 LuxR-AHL Fluorescence 485-535 absorbance related to time.
Fig.12 RpaR-AHL Fluorescence 485-535 absorbance related to time.
Compared between different quorum sensing systems, the Lux quorum sensing system has lower background leakage, higher orthogonality and robustness, and better expression stability. Therefore, we choose Lux quorum sensing system as the input signal controller for node A.
The Repressive RNA Switch pT181 is the extra regulator of our Negative Feedback Loop (NFBL). We are utilizing pT181 attenuator – a dual control repressors – to regulate both gene transcription and translation in a fast and robust way. We have submitted it as our improved part since it increases repression from 84% to 98% compared with that of Kyoto 2013 submitted. As RNA transcriptional regulators are emerging as versatile components for genetic network construction, we believe that improving the part in this library is essential for advancing synthetic biology. We hope our improvement of pT181 to iGEM parts will encourage future teams to implement this versatile, highly orthogonal, and effective regulator in their circuits.
Key achievement
- Characterization of pT181-mediated target gene activation in various conditions.
- Improvement of a new sense target sequence of pT181 attenuator.
- Update of the BioBrick Registry library by improving a RNA-logic toolset.
Overview
Our engineered cells need a Three-Node Negative Feedback Loop to construct a more sensitive and high-fidelity control system. And pT181 is the part that plays the role of repressor in this loop.
pT181 is a part composed of a sense target sequence and an antisense RNA that can regulate gene transcription and translation. Residing in the 5΄ untranslated region of the target gene, it can regulate the expression of a downstream gene at both transcriptional and translational levels.
At transcriptional level, the anti-terminator, which is a part of the sense target sequence will anneal with the 5’ region of the terminator stem without antisense RNA. In this case, the terminator could not format, which means the RNA polymerase can start transcription. At translational level, in the absence of antisense RNA, a ribosome binding site (RBS) for the gene of interesting is exposed, so that the ribosome could bind to it to begin translation.
While the antisense RNA is present, the formation of terminator will be allowed as the anti-terminator is sequestered due to the kissing hairpin interaction between the antisense RNA and the sense target sequence. In this way, the downstream transcription will be prevented. As for the translational level, the occlusion of the RBS by the terminator hairpin will prevent the translation of the target gene.
In conclusion, while antisense RNA is not present, the gene will express as normal, while antisense RNA is produced, the downstream target gene will be repressed effectively.
The RNA regulators show sufficient advantages over traditional protein-based regulatory systems, including:
- Programmability: As Watson-Crick base pairing is predictable, the RNA-RNA interaction can be predicted by sophisticated software tools. In this way, a RNA switch can be designed artificially, which are difficult for proteins.
- Lower metabolic cost: Compared with proteins, the RNA switches dispense with translation step, which saves a great amount of resources.
- Fast response: RNA switches could propagate signals faster than proteins considering the fast degradation rates of RNAs.
Despite these advantages, RNA regulators still suffer from incomplete repression in their OFF state, making the dynamic range less than that of the proteins. This leak can cause the network to function incorrectly. Therefore, we submit the dual-control pT181, which can solve this problem.
The dual-control pT181 we submitted offers a significant advantage over previous iGEM parts that submitted in 2013:
- Reduce leak: As our pT181 attenuator could regulate both transcription and translation in a single compact RNA mechanism, which means it could provide stronger functions without increasing burden. This dual control repressor is able to increases repression from 85% to 98%.
Our approach
pT181 attenuator is the part C in our negative feedback loop. As the regulator in our system, pT181 will be activated by part B, since we place part B’s target sequence upstream the gene for pT181 antisense. Meanwhile, pT181 will repress the part A due to the pT181 sense target upstream the gene for part A. Consequently, the upregulation of part B caused by the expression of part A will indirectly repress part A. In this case, the whole system is ready to response change of the input signal.
In the absence of input signal, we hope that there is no expression of the output. However, due to the inevitable leak from the part A and part B, it seems to be impossible to avoid expression of the output. With pT181 in our system, we could avert it at utmost because of the efficient repression of it – just tiny quantity of pT181 will prevent most of the leak. This ensures the minimum expression of the output without input signal.
When the input signal is present, it will induce the expression of part A, which will upregulate the part B a lot. In this way, the output will be strongly expressed. But the upregulation of part B would cause the expression of pT181, which will repress the part A. And this will cause the low expression of part A and indirect low expression of part B. Although the remains in the environment would keep the amount of output, the low quantity of each part of the system will prepare it for any change from the input signal. In this case, the output of the system would response to the change of the input in a very short time. This can also eliminate the possible superposition between outputs from different input signals.
We used our model to predict whether the high repression effect of pT181 will cause the silence of the output, as it may cause the silence of part A. However, our model shows that the output will respond to the input perfectly, which support our experiment a lot.
In conclusion, the presence of pT181 attenuator will reduce leak of our system at utmost, as well as allowing it to rapidly respond to the changing signal.
Experimental design
Fig.13 A schematic representation of the experimental group plasmid. This has the basic pT181 Antisense under control of a constitutive promoter, as well as a GFP gene downstream of the pT181 sense target under the control of a constitutive promoter.
Fig.14 A schematic representation of the positive control plasmid with the GFP gene downstream of the pT181 sense target under the control of a constitutive promoter, without a pT181 Antisense on it.
We generated two plasmids, one is for experimental group, the other is for a positive control. The experimental plasmid contains the antisense sequence downstream of a constitutive promoter and followed by a double terminator on a high-copy plasmid. Meanwhile, there are also a GFP gene with a ribosome binding site downstream of the pT181 sense target sequence. The GFP coding sequence is also downstream of a constitutive promoter and followed by a double terminator. The positive control plasmid contains the same as the experimental plasmid except for the antisense sequence.
We did two groups of pT181 expression experiments. First, as a part that needs to show strong inhibition, we should ensure that its inhibitory effect is obvious enough. Therefore, we compared experimental group, positive control and negative control，which is transformed into a normal GFP plasmid. Depending on the GFP expression, we can prove that our work has a high credibility. Additionally, in the group above, three types of flora from Interlab are cultivated for contrast. The aim is to compare the statistics of pT181 and verified Interlab to find out which repressor level pT181 is in when put into practical application. What’s more, for different temperatures, we perform another experiment. Under 30℃ or 37℃, we cultivate the same flora and compare the difference of fluorescence from standardization flora. In this way, we can explore the impact of temperature.
For the first group, we transform different plasmids into the E. coli in the tubes and cultivate for hours (37℃, 220RPM). Then we used ELISA plate to detect the change of fluorescence and OD600 over time. What should be noticed is that we set the original flora at OD600=0.05 to guarantee flora proliferating at the same concentration. As the repression of pT181 attenuator is so powerful that the fluorescence of the experimental group is hard to detect. As a result, to remove LB medium’s fluorescence background, we centrifuge fluid, discard supernatant, add PBS buffer and resuspend before detection.
For the second group, our purpose is to study the function of pT181 under different temperatures. The reason why we cultivate bacterial under 30℃ or 37℃ is that function of RNA parts is greatly influenced by thermodynamics parameters (such as temperature). We also guaranteed the same start concentration as the first one. When plateau period arrives, we detect fluorescence and OD600.
Result
Fig.15 400x image of positive control under fluorescence microscope.
Fig.16 400x image of experimental group under fluorescence microscope.
Reference: Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator
Westbrook, Alexandra M ; Lucks, Julius B
Nucleic acids research, 19 May 2017, Vol.45(9), pp.5614-5624