Revision as of 20:36, 20 October 2019

AraC-Pc-pBAD-RBS1-Colicin E2-T500-T1/TE-MicCsRNA-PA1/O4

This composite part is designed to insert the toxin Colicin E2 into the bistable system.

Presenting with BBa_K3202066-BBa_K3202067, this part is to test the function of the toxin within our bistable system.

Bistable System Module

I.Background

We are inspired by a lately published article written by Belén Calles, Angel Goñi-Moreno, and Víctor de Lorenzo to design a double-bistable system which achieves zero basal expression of gene of interest in the absence of any inducer while ensuring constant transcriptional capacities and induction rates among promoters.

“The mechanism of a bistable system is elaborated below: a repressor transcriptionally inhibits the promoter which is responsible for the transcription of sRNA, while the sRNA translationally inhibits the repressor. The gene of interest is translationally coupled to the repressor gene. That is to say, the repressor protein R targets the strong promoter which the inhibitory sRNA is produced, so that R and sRNA are mutually inhibitory. Such arrangement helps to minimize the basal activity of the inducible module and thus suppress leaky gene expression levels.”

II.Design

In our system(Fig. 1), we have transcriptional repressors R1 and R2 expressed through the inducible promoter but translationally inhibited by the MicC sRNA1 and sRNA2 respectively, since RBS2 and RBS3 is directly inhibited by sRNA while they initiates the transcription of the two repressors; the gene of interest is translationally coupled to the repressor gene while three promoters which produces the inhibitory sRNA are targeted by their corresponding repressors. Therefore, repressors and sRNAs are mutually inhibitory.

Figure 1

RBS1

The control of the recombinase in coordination with the upstream repressor gene, which is the target of the sRNA, requires a translational coupler cassette. “An efficient mechanism to reach this goal is based on the ability of translating ribosomes to unfold mRNA secondary structures. In our design, translational coupling is achieved by occluding the RBS of gene of interest by formation of a secondary mRNA structure, containing a His-tag sequence added to the 3 ́end of the repressor gene. The hairpin structure prevents the ribosomal recruitment to the recombinase and therefore its translation. In contrast, when the upstream repressor mRNA is actively translated, the 70s ribosome disrupts inhibitory mRNA secondary structure in the downstream gene translation initiation region thus allowing its expression.”

However, during the experiment we tested that the RBS1 (Fig. 2) sequence provided by the original article fails to function in our circuit, we found RBS2 (Fig. 3) with a different working mechanism. The structure of AUGA in RBS2 acts as both the stop codon (AUG) of the former gene sequence, releasing the former peptide chain; takes a step (gene code) backward, pinpointing a new ribosome binding site, and initiated the translation of next gene sequence with the start codon (UGA). However, finally we still used RBS1 since the crux actually lied in another place.

Figure 2

Figure 3

sRNA

Small non‐coding RNAs (sRNAs) have important functions as genetic regulators in prokaryotes. “sRNAs act post‐transcriptionally through complementary pairing with target mRNAs to regulate protein expression. Antisense sRNAs negatively regulates proteins by destabilizing the target protein's mRNA. Antisense sRNAs prevent translation by binding to the target mRNAs in a process mediated by the RNA chaperone Hfq.” On binding, both the mRNAs and sRNAs are degraded, suggesting that prokaryotic sRNAs—unlike their eukaryotic counterparts—act stoichiometrically on their targets.

Repressor

“A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA.”

We assume that when the whole system is applied to a factory, the situation where inducer is present is in the working condition of the bacterium. Within one single layer of the bistable system, the transcriptional factor is turned on, Pm initiates the expression of R1, which raises the concentration of R1 and thereby exerts greater inhibition on PR1 and therefore decreases the concentration of sRNA1. As sRNA1 is inhibited the inhibitory effect it exerts on R1 accordingly decreases, therefore the recombinase is expressed through the first layer, and vice versa. Nevertheless, given that the expression of R1 inhibits PR1, even if the recombinase is expressed PR1 can be flipped over but cannot express the second layer. Since there is no further input signal to the second layer of the bistable system, the inhibitory effect of sRNA2 dominates over R2, therefore, both R2 and the toxin cannot be expressed. That is to say, the additional second layer ensures that there is no leakage of toxin when inducer is present.

However, when the bacterium is stolen and thereby inducer isn’t present, the transcriptional factor is turned off, promoter Pm doesn’t work. Therefore, sRNA1 dominates over R1 and then the recombinase is not expressed in the first layer. Since there is a decrease in the expression of R1, the inhibitory effect it exerts on PR1 decreases, therefore PR1 which has already been flipped over when inducer is present (working condition) initiates the expression of R2. Applying the same logic, finally R2 dominates over sRNA2 and expresses the toxin to kill the cell. (Fig. 4)

Figure 4

Why double layer?

The reason why we designed such double-bistable system can be attributed to the consideration that the double-decked bistable systems of both recombinase and toxin could further reinforce the insurance of zero expression of toxin.

When the second bistable system coupling the toxin with the first layer of recombinase, double insurance of zero expression of recombinase and toxin is achieved. However, as the diagram shown below, a single bistable system where toxin is coupled without the second layer of insurance could collapse if the mutual inhibitory effect of repressor and sRNA within the first layer malfunctions. Whereas such concern could be eliminated by the second layer of toxin with a bistable system around.

III. Experiment & Results

After the circuit (Fig. 5) including only one layer of the bistable system is constructed with recombinase being replaced by sfGFP, LacI & TetR serving as repressor, and used 3MBz & AraC as inducers, we qualitatively tested the fluorescence of the reporter protein under two situations when excited blue light. Theoretically, sfGFP should be expressed when inducer is present since LacI, which transcribes sfGFP, dominates over the inhibitory force of sRNA. However, in the experiments when inducers are present and absent, there is no fluorescence of sfGFP (BN027/Contains BBa_K3202074 & 28/Contains BBa_K3202075 & 29/Contains BBa_K3202076 & 30/Contains BBa_K3202077). We deduced that there must be defects lying in our circuit.

Figure 5

Figure 6 A: BN006/Contains BBa_K3202029 (without GFP) when inducer is absent and present; B: BN007/Contains BBa_K3202030 (AraC + sfGFP) when inducer is absent and present; C: BN027/Contains BBa_K3202074 (AraC + LacI + sfGFP) when inducer is absent and present; D: BN029/Contains BBa_K3202076 (AraC + TetR + sfGFP) when inducer is absent and present

Figure 7 A: BN006/Contains BBa_K3202029 (without GFP) when inducer is absent and present; B: BN007/Contains BBa_K3202030 (Xyls + pBAD + sfGFP) when inducer is absent and present; C: BN028/Contains BBa_K3202075 (Xyls + LacI + sfGFP) when inducer is absent and present; D: BN030/Contains BBa_K3202077 (Xyls + TetR + sfGFP) when inducer is absent and present

Figure 8: As shown in the graph from flow cytometry experiments: in the presence of a fixed concentration of inducer (1.0 mM of Ara) at the indicated time points (5 to 240 min after induction), BN006/Contains BBa_K3202029, which carries no sfGFP, has low fluorescence level under 100 a.u. being expressed in inducer’s presence. BN027/Contains BBa_K3202074 (under the control of bistable system) shows similar pattern as time increases as that of BN006/Contains BBa_K3202029. BN007/Contains BBa_K3202030 (without control of bistable system) showed higher fluorescence level throughout the experiment

Figure 9: As shown in the graph from flow cytometry experiments: at same time period, as the conc. of Ara increases from 0 to 1.0 mMol, BN006/Contains BBa_K3202029 (no sfGFP) and BN027/Contains BBa_K3202074 (under the control of bistable system) showed both fluorescence level around 100 a.u., revealing a lack of expression of sfGFP; while BN007/Contains BBa_K3202030 displayed increase in fluorescence level as inducer concentration increases

Analyzing through the whole circuit, we deduced that the crux might lie in RBS1 for the sake of its overwhelmingly strong hair-pin structure that even if the 3’ end of LacI is translated the ribosomes produced still couldn’t melt sRNA secondary structure, thereby sfGFP cannot be translated. Therefore, we replaced RBS1 with a commonly used RBS: B0034 with weaker strength in order to ensure that there is no problem lying in other sites in the circuit. We tested the sfGFP’s fluorescence in inducers’ presence under excitation of blue light. The expression of sfGFP in the second attempt (Fig. 10B, Fig. 11B & D) proved to us that there’s nothing wrong with the rest of the circuit except for RBS1.

Figure 10 A: BN040/Contains BBa_K3202047 (Xyls + TetR + RBS B0034 + sfGFP) when inducer is present and absent; B: BN009/Contains BBa_K3202031 (Xyls + msfGFP) when inducer is present and absent

Figure 11 A: BN007/Contains BBa_K3202030 (AraC + pBAD + sfGFP) when inducer is absent; C: BN007/Contains BBa_K3202030 (AraC + pBAD + sfGFP) when inducer is present; B: BN036/Contains BBa_K3202046 (pBAD + LacI +RBS B0034 + sfGFP) when inducer is absent; D. BN036/Contains BBa_K3202046 (pBAD + LacI +RBS B0034) + sfGFP when inducer is present

Figure 12: As shown in the graph from flow cytometry experiments: at same time period, in the absence and presence(1mMol) of inducers, both plasmids (BN036/Contains BBa_K3202046 & BN040/Contains BBa_K3202047) have their fluorescence level 10 times lower in inducer’s absence than that in inducer’s presence

Through further searching we found that RBS2 has a weaker hair pin structure while it could support the translation of repressor. We replaced RBS B0034 with RBS2. Results are shown in the two graphs below. Plasmid with RBS2 expressed sfGFP with quite a high expression leakage when both inducers are absent. We thought that such high expression leakage might be the result of the weak hair-pin structure of RBS2, which means that we still need a strong RBS to reduce expression leakage. Hence, by we were inspired to realize that we might wrongly include the terminator of LacI before RBS. If we include the terminator, the transcription of LacI stops there and therefore the LacI protein produced by translation ends in front of RBS and leaves the mRNA secondary structure before melting it, sfGFP failed to be expressed.

Figure 13 A:BN009/Contains BBa_K3202031 (Xyls + sfGFP) when inducer is absent and present; B:BN045/Contains BBa_K3202049 (TetR + Xyls +RBS2) when inducer is absent and present; C:BN049/Contains BBa_K3202050 (LacI+ Xyls + RBS2) when inducer is absent and present

Figure 14 A:BN006/Contains BBa_K3202029 (without GFP) when inducer is absent and present ; B:BN043/Contains BBa_K3202048 (pBAD + LacI +RBS2 + sfGFP) when inducer is absent and present; C:BN007/Contains BBa_K3202030 (AraC + pBAD + sfGFP) when inducer is absent and present; D:BN051/Contains BBa_K3202051 (pBAD + TetR + RBS2 + sfGFP) when inducer is absent and present

Figure 15

Figure 16

As shown in Fig. 15 & 16 from flow cytometry experiments: at the same time period, the conc. of inducer increases from 0.000 to 1.000 mMol, BN006/Contains BBa_K3202029, which carries no sfGFP, has a low fluorescence level around 0.60 a.u. in inducer’s absence. BN007/Contains BBa_K3202030 and BN009/Contains BBa_K3202031 without the control of bistable system had their fluorescence value at a relatively higher level in inducer’s absence and their fluorescence value increase as conc. of inducers increases. BN043/Contains BBa_K3202048 & 51/Contains BBa_K3202051 & 45/Contains BBa_K3202049 & 49/Contains BBa_K3202050 (under the control of bistable system) have their fluorescence value lower than that of BN007/Contains BBa_K3202030 and BN009/Contains BBa_K3202031 in inducer’s absence and have their fluorescence value as conc. of inducer increases.

Figure 17

Figure 18

As shown in Fig. 17 & 18 from flow cytometry experiments: in the absence of inducer at the indicated time points (4h and 24h after induction), BN006/Contains BBa_K3202029, which carries no sfGFP, has a low fluorescence level around 0.60 a.u. in inducer’s absence. BN043/Contains BBa_K3202048 & 51/Contains BBa_K3202051 & 45/Contains BBa_K3202049 & 49/Contains BBa_K3202050 (under the control of bistable system) have their fluorescence value higher than that of BN006/Contains BBa_K3202029 while lower than that of BN007/Contains BBa_K3202030 &BN009/Contains BBa_K3202031 (without the control of bistable system) in inducer’s absence.

We improved our gene circuit upon that reflection, with exclusion of the terminator of repressors and replacement of BRS2 using RBS1, and made the third trial. The results are shown below.

Figure 19 A: BN007/Contains BBa_K3202030 (AraC + pBAD + sfGFP) when inducer is absent and present; B: BN055/Contains BBa_K3202045 (Xyls + TetR + RBS1 + sfGFP) when inducer is absent and present; C: BN054/ Contains BBa_K3202044 (AraC + LacI +RBS1 + sfGFP) when inducer is absent and present

As shown in Fig. 20 & 21 from flow cytometry experiments: in the absence of inducer at the indicated time points (4h and 24h after induction), BN006/Contains BBa_K3202029, which carries no sfGFP, has a low fluorescence level around 0.60 a.u. in inducer’s absence. BN007/Contains BBa_K3202030 and BN009/Contains BBa_K3202031are two plasmids without the control of bistable system; they displayed high fluorescence level as time passed. BN054/Contains BBa_K3202044 and BN055/Contains BBa_K3202045 have similarly no fluorescence level as BN006/Contains BBa_K3202029.

Figure 20

Figure 21

As shown in Fig. 22 & 23 from flow cytometry experiments: at same time period, as the conc. of inducers increases from 0.0001 (nearly no inducer) to 10 mMol, BN006/Contains BBa_K3202029, which carries no sfGFP, always has a low fluorescence level around 0.60 a.u. While both two plasmids (BN054/Contains BBa_K3202044, induced by Ara; BN055/Contains BBa_K3202045, induced by 3MBz) have their fluorescence level increase from around 10 a.u. (lack of expression of sfGFP) when inducer is absent to a higher value.

Figure 22

Figure 23

Toxin Module

I.Background

Toxin proteins are good materials for detecting leakage of inducible promoters. Because sometimes a molecule of translated toxin, it can have a great impact on cell growth. To further test the leakage effect, we incorporate toxin protein—Kid, ParE and Colicin—into the plasmid. ParE toxins are DNA gyrase (Gyr) inhibitors.Toxin Kid which closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli can inhibit DNA replication. Toxin Colicin binds to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarization of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis.

II.Design

We chose pBAD as the inducible promoter in this experiment for it has lower basal leakage level than Xyls. We set all together four different group including one control. We measured OD 600 value which indicate cell growth for the following seven hours.

III.Experiment & Results

At the first level of experiment we incorporate toxins with pBAD & AraC. At the absence of inducer, all of the three toxin experiment group demonstrated a lower OD value with more significant low OD value in BN062/Contains BBa_K3202052 (with toxin ParE) and BN065/BBa_K3202054 (with toxin Colicin) than control. Cell growth was affected in each experimental group. When measured after 12 hours, the trend is more obvious with BN065/BBa_K3202054 and BN062/Contains BBa_K3202052 demonstrate a OD value of 0.1891 and 0.1604 respectively. Whereas control group is 0.7755. The result suggests that even without inducer, pBAD system have noticeable leakage when incorporated with toxins which may affected cell growth in some cases.

Figure 1: Growth Curve for Toxin plasmid with pBAD system in the period of 7 hours.

Figure 2: Bar graph of OD 600 value for Toxin with pBAD system after 12 hours.

We want to test our bi-stable system to see wether it can really prevent leakage. We construct three toxin-bistable-system plasmid.When compared to BN063(Kid&pBAD) during a seven hours period, we can observe a significant improvement in cell growth in BN084 as OD 600 value have is higher. This suggests that our system has a significant role in inhibiting the expression of toxin proteins. Later, when we inoculated the colonies, we found that the toxin protein integrated into the bistable state was significantly inhibited in expression. Cell growth was significantly better than BN063.

Figure3: Growth curve for BN063(Kid&pBAD) and BN084(Kid&bi-stable system) in the period of 7 hours.

Figure 4: Bar graph of OD 600 value for BN063 and BN084 after 12 hours.

Figure 5:Colony growth for three types of toxin-bistable-system after 20 hours

Sequence and Features