Difference between revisions of "Team:Imperial College/Results"

The comparator module is the central processing unit of our Genetically Engineered Artificial Ratio (G.E.A.R.) system. We are utilising STAR1, short for Small Transcriptional-Activating RNA, a novel RNA-logic technology. It is our best basic part and a completely new addition to the BioBrick Registry, allowing for rapid and robust regulation of gene transcription. RNA-based logic systems have proven to be very popular over the past year, and we believe that expanding this library of tools is essential for advancing synthetic biology. We hope that our design will encourage future iGEM teams to implement STAR in their circuits as a versatile, highly orthogonal, and effective method of gene regulation.

Key Achievements

• Characterisation of STAR-mediated target gene activation in various conditions.
• Designing of a new anti-sense RNA sequence complementary to STAR, the Anti-STAR.
• Characterisation of quorum-sensing regulated STAR and Anti.STAR.
• Expansion of the BioBrick Registry library by addition of a new RNA-logic toolset.

Our engineered cells need a way to compare the the population sizes of all the different cell species in a co-culture. Therefore, we designed a comparator module, which compares the the amount of each type of quorum signal (AHL) coming from the communication module and regulates gene expression in the growth module in response.

STAR is is the first instance of small bacterial RNAs (small RNAs) being used to directly activate transcription. STAR requires a sense target sequence RNA (the pAD1 plasmid attenuator sequence) fused upstream of the regulated DNA sequence gene (in this case the growth module). The STAR-target forms an intrinsic hairpin structure, preventing RNA polymerase mediated elongation of transcription. Thus it acts as a terminator of transcription. However, when STAR is produced, it interacts with the target sequence and relieves the hairpin formation, allowing transcription to continue.

To regulate STAR activity, we designed an anti-sense RNA sequence (Anti-STAR). The Anti-STAR sequence is complementary to STAR, however represents only part of the sense target RNA (pAD1 attenuator sequence). When transcribed, Anti-STAR sequesters STAR, preventing it from interacting with the target sequence to activate transcription.

Our STAR approach offers significant advantages over traditional protein-based regulatory systems.
These include:
→ Low metabolic burden- transcription of the short 68 nucleotide sequence avoids energy-intensive translation steps.
→ Portability- trans-acting RNA based regulation is found throughout bacterial kingdom and is not host-specific.
→ 96-fold activation: the pAD1 plasmid sense attenuator and anti-sense STAR sequences are highly orthogonal.
→ Robustness- Watson-Crick base pairing avoids need for 3D structural protein-protein interactions.
→ Very fast and precise signal propagation controlled by quick RNA degradation rates- reducing lags in circuit.

We used one quorum regulator to control the transcription of STAR in response to the amount of quorum signal produced by one of the populations; and a second quorum regulator to control the transcription of Anti-STAR in response to the amount of quorum signal produced by a second population. The balance between the amount of STAR produced and the amount of Anti-STAR produced determines the amount of transcription of the gene downstream of the target sequence. Therefore, the relative population sizes can be used to control the expression of the growth module

Let’s assume that we have two different cell populations in co-culture: Cell A and Cell B. Each cell population synthesizes one type of quorum signal (AHL). Cell A synthesises AHL A and Cell B synthesises AHL B. In Cell A, the quorum regulator that controls transcription in response to AHL A is used to control the transcription of STAR. The quorum regulator that controls transcription in response to AHL B is used to control the transcription of Anti-STAR. These roles are reversed in the Cell B population (AHL B controls the transcription of STAR and AHL A controls the transcription of Anti-STAR).

When both population sizes are the same:

• Equal amounts of STAR and Anti.STAR are transcribed in both cells.
• They sequester each other.
• The negative growth regulating protein is not transcribed, or expressed in either population.

• In the cell with the higher population size, more STAR is transcribed than Anti-STAR.
• More of the negative growth regulatory gene is expressed.
• This slows down growth rate of the faster growing population, allowing the slower population to catch up in size.

We generated a two-plasmid system for characterisation experiments.
The first plasmid (Figure 1) contains the STAR sequence downstream of a constitutive Anderson promoter and followed by the t500 transcriptional terminator on a high-copy plasmid. When the STAR RNA is transcribed, it binds to the 5’ stem of the target sequence in trans, preventing terminator hairpin formation and allowing RNAP transcription elongation of the downstream sequence.

Figure 1: A schematic representation of the first plasmid. This has the basic STAR part (STAR-t500) under control of the constitutive promoter j23119.

The second plasmid (Figure 2) is a reporter plasmid that contains the superfolder GFP (SFGFP) gene with a ribosome binding site immediately downstream of the STAR-target (pAD1 plasmid attenuator) sequence. The SFGFP coding sequence is under the control of a constitutive Anderson promoter and also has its own TrrnB terminator. The STAR-target forms an intrinsic terminator hairpin structure, facilitating RNA polymerase (RNAP) falloff at the beginning of the mRNA and thus prevent transcription of the downstream coding sequence. In the presence of STAR this hairpin structure is alleviated, allowing transcription to resume.

Figure 2: A schematic representation of the reporter plasmid with the coding sequence of superfolder GFP (SFGFP).

The experiments that we have conducted are:

1. Characterisation of STAR target repression (activation of SFGFP) at 300C and 370C
2. Characterisation of the activation range of STAR under control of pLas and pRhl (awaiting data)
3. Characterisation of the deactivation range of STAR when both STAR and Anti-STAR are induced by varying concentrations of Las and Rhl AHLs (awaiting data)

Figure 3(i): Characterisation of STAR target repression (activation of SFGFP). Fluorescence assay.

Figure 3(ii): Characterisation of STAR target repression (activation of SFGFP). Fluorescence/ absorbance data at 100 minutes(SFGFP).

All characterisation was done in E. coli Top 10 cells. Each test contained three test replicate cultures of a single colony. Both graphs show vertical error bars representing Standard Deviation.The autofluorescence background control used is E. coli DH10 cells with no reporter plasmid.

Both the experimental tests have a two-plasmid system:
The negative control test has the sense target-SFGFP reporter plasmid along with just a constitutive promoter on a separate plasmid. The positive test has the sense target-SFGFP reporter plasmid as well as the STAR-antisense RNA under control of the constitutive promoter on a different plasmid. The fluorescence and absorbance values for experimental negative control and positive test were first corrected by subtracting the average of each of the three corresponding readings for the control E. coli Top 10 cells. The Fluorescence/Absorbance (F/Abs.) value of the experimental negative control and positive test were initially calculated and divided by the average F/Abs. of the control E. coli Top 10 cells.

• Characterise the deactivation curve of STAR by having AHL A-STAR and AHL B- Anti-STAR on the same plasmid.
• Run all the characterisation experiments by using Las and Cin as the two bi-directional orthogonal quorum sensing systems.

We attempted to ligate STAR and Anti-STAR using BioBrick assembly method a number of times, each time optimising various steps in the protocol and having failed each time. We think that perhaps a recombination event is occurring in the cells as the STAR composite and Anti-STAR composite sequence are very small and both contain the constitutive Anderson promoter j23119. The assembly of the quorum-sensing regulated STAR and Anti-STAR were fairly easy on their own. In future, The star and Anti-STAR could be synthesized en-bloc as a construct in itself for the characterisation of Anti-STAR sequestering STAR.

The growth module is the last of the three modules constituting our Genetically Engineered Artificial Ratio (G.E.A.R.) system. If the STAR system determines that one population's quorum signal is too high, it needs to activate transcription of a protein that arrests the growth of that population. We investigated a range of growth-regulation targets, including the established techniques of antibiotics and auxotrophy. We decided on a novel growth regulatory system, utilising the phage gene Gp2, and the growth regulation achieved is rapid, effective and reversible, and has many advantages over existing growth control methods.

• Characterised a novel, effective and reversible growth control method for E. coli.
• Improved the pBad-AraC construct by adding a reverse terminator.
• Characterised our pBAD-AraC construct in top10 cells.

The final part of our circuit is the growth-regulation system. This needs to be a protein that inhibits the growth of the cell when its expression is activated by STAR. Our target gene had to have a number of features in order to prevent the system from losing balance and to minimise lag time. It had to inhibit growth rapidly without killing the cell, and allow the cell to recover normal growth conditions after the STAR system no longer activates the circuit.

We settled on four target genes:

1. Cat, encoding Chloramphenicol acetyltransferase, which is an enzyme that confers resistance to the antibiotic chloramphenicol. We decided to use chloramphenicol because it is a bacteriostatic rather than a bactericidal antibiotic. When using Cat, chloramphenicol would be added to the growth medium.
2. LeuB - an enzyme in the biosynthetic pathway of leucine in E. coli- the gene has been used before as a control method in co-culture. When using LeuB, we would need to use a strain that is auxotrophic for leucine and a growth medium lacking leucine.
   
   Under the conditions described above, expression of LeuB and Cat would both be required for normal growth, and so in the context of our system, growth inhibition would be achieved by STAR triggering production of a protein that inhibits their expression, e.g. using an inverter.
3. Gp2 is a gene from the E. coli bacteriophage T7 phage, which slows down cell growth by binding reversibly to the E. coli RNA polymerase complex, thus inhibiting transcription. T7 phage infection is characterised by the hindrance of bacterial growth, and Gp2 has been suggested as a potential antimicrobial agent.
4. Gp0.4 is another T7 phage gene which inhibits growth by binding to the FtsZ ring during mitosis, preventing cytokinesis (the final stage of cell division where the two daughter cells separate).

To characterise these genes, we placed them under the control of the arabinose-inducible pBAD promoter. This was done because constitutive expression of growth-inhibiting proteins would have made characterisation extremely difficult. After several attempts at cloning, we were able to obtain pBAD-Gp2 and pBAD leuB constructs. Our characterisation efforts then focussed on Gp2 because it allows direct inhibition of growth without the need for an inverter.

Gp2 is also an ideal protein for our purposes because:

• It has a small coding sequence (>200bp) and therefore it is transcribed faster than LeuB or Cat, which both have a coding sequence of about 1kb.
• Gp2 does not require the use of an inverter, which would slow down the circuit significantly. Therefore Gp2 is faster than both LeuB and Cat, as they require the implementation of an inverter to fit in our G.E.A.R. system.
• Auxotrophy systems, such as the LeuB system, require the creation of knockout strains. Gp2 does not depend on any auxotrophy system and it can therefore be used directly on the species of interest. This could potentially simplify the building step and minimize the costs of iGEM teams projects.
• Using chloramphenicol resistance reduces the amount of plasmids the cell can be transformed with, and also causes issues with regards to antimicrobial resistance. On the contrary, Gp2 does not cause any of those problems.
• Cat and LeuB both require special media conditions, reducing the versatility of the system. Gp2-based system does not need any special medium to function properly, thus making it easier to work with.



These reasons, identified during our reflexivity process, lead us to choose Gp2 as our growth regulating protein in our G.E.A.R. system. Reflexivity is the core aspect of our integrated human practices, that has helped us identify problems and solutions through our project. For more information on our reflexivity process, please visit our integrated human practices page!





Experimental Design


We characterised the activity of the araBAD operon by placing a GFP reporter under control of the pBAD promoter, and quantifying the amount of fluorescence produced by varying concentrations of arabinose. Once we had obtained an activation range, we moved on to characterising the activity of Gp2.

For all characterisation experiments, transformed cells were grown from stocks overnight, and subcultured in the morning. After three to four hours, (when the cultures were in exponential phase) the cultures were diluted to 0.05 OD and aliquoted into a 96 wells plate, then treated with varying concentrations of arabinose. Repression experiments involved treating the culture with arabinose for two hours, before aliquoting into a plate and adding a range of concentrations of glucose.





Results


Our results show that Gp2 effectively inhibits E. coli growth in a reversible manner. At the highest level of induction on the high copy psB1A2 plasmid, the cell can recover from the growth inhibition on its own after approximately eight hours, but this was not observed at lower induction strengths. Our results also indicate that once Gp2 production is switched off, the cells can recover back to a normal growth rate within an hour.





Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. (BBa_K1893017).Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 OD and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (OD600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation





Figure 2: Transfer function of normalised GFP fluorescence against concentration of L-arabinose





Figure 3: Growth inhibition of Top10 cells by induction of Gp2 by L-arabinose. Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 OD and inoculated with L-arabinose at the 0 minute timepoint. O.D. was recorded at 600nm. Reported values represent the mean normalised O.D. for three repeats, with error bars representing standard deviation.





Figure 4: Recovery of growth by Top10 cells after arabinose-induced pBAD operon was switched off by glucose-mediated catabolite repression. 100uM of L-arabinose was added to the culture 2 hours before the 0 minute timepoint and D-glucose was added at the 0 minute timepoint. . pBAD-GFP controls indicate that the pBAD operon was switched off at approximately after approximately 250 minutes. Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 OD, which was recorded at 600nm. Reported values represent the mean normalised O.D. for three repeats, with error bars representing standard deviation.

What’s Next?

• Investigate how long it takes for E. coli to escape the growth repression by Gp2.
• Investigate how to prevent cheater cells (mutants who inactivate the growth control circuit), potentially by creating an operon containing Gp2 and an essential metabolic enzyme.
• Gp0.4 was abandoned because of ligation issues, but further work would include an attempt to characterize it again. Gp0.4 has an advantage over Gp2 in that it doesn't affect protein production, and so will not unbalance the circuit (Gp2 will also prevent production of proteins that are necessary for the circuit to function). However, it does appear that Gp2 plays a potential role in damping the ratio oscillations in our circuit (modelling link).



Experience


It took several attempts to clone genes into the pBAD construct, and we were unable to successfully insert two of four of our genes into the pBAD construct. We had some success with increasing the insert:vector ratio (NEB). Gp2 was extremely easy to work with, and worked as expected first time.

Note: Growth of BY4741 at 37°C was included but infection after inoculation has likely occurred.