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vsw-3 rnap

1. Usage and Biology

Enabling low-temperature-induced expression was one of the needs for our anti-icing program. Among the numerous methods for regulating gene expression in bacteria, transcriptional control is the most widely developed and implemented (1). Even though applications of thermogenetics have come of age (2) , many methods for gene regulation are based on post-transcriptional control due to the intrinsic properties of RNA while most transcriptional control methods are always developed on the engineering of widely-used and mature systems, such as T7 RNA polymerase (T7 RNAP) and λ-CI repressor. Here, inspired by the gene resources mining of extremophiles (3),(4), it is the first time to introduce a cold-adapted RNA polymerase, VSW-3 RNA polymerase from the chillophilic phage VSW-3 of from a plateau lake (5), to construct a novel low-temperature-induced prokaryotic transcription-activating system and further apply in building AND-logic gate for tight control of gene expression. The VSW-3 RNAP is a novel single-subunit RNA polymerase encoded by the chillophilic phage VSW-3, which was first characterized in vitro in 2022. VSW-3 RNAP showed a good low-temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript in vitro (5). Moreover, the in vitro transcription products of VSW-3 RNAP were used to prepare mRNA for mRNA therapy in vivo due to the superior protein expression levels of VSW-3 RNA transcripts, compared to T7 RNAP transcripts (6). Based on the previous work, we believed that VSW-3 RNAP would also perform well in vivo for the purpose of developing a new transcriptional control system. As a cryophilic phage polymerase, VSW-3 RNAP is expected to be more active at low temperatures, so we determined to characterize VSW-3 RNAP and its cognate promoter(s), subsequently tried to apply the novel expression system in logic gate construction and recombinant protein production as a coldness-inducible pattern, all of which will be described and recorded in the following document.

2. The exploration of available promoters

2.1 Introduction

Similar to the T7 system (T7 RNAP and its cognate promoter pT7), VSW-3 RNAP should also have a specific cognate promoter in the genome of the VSW-3 phage. However, only a few promoters appear on the genome of VSW-3 phage, so the prediction of its promoters is difficult. The in vitro transcription system established on the VSW-3 RNAP has focused on the identified 18-bp promoter sequence 5′-TTAATTGGGCCACCTATA-3′, which was part of the 21-bp predicted one 5′-TTAATTGGGCCACCTATAGTA-3′ (5). We named the 18-bp promoter pVSW-3 then constructed the reporting circuit pVSW-3-B0034-gfp-B0015, followed by co-transforming the circuit with L-arabinose-induced VSW-3 RNAP-expressing plasmid (BBa_K4907114_pSB1C3) into E. coli BL21(DE3) and characterizations. However, no significant difference was observed between the groups with and without expressing VSW-3 RNAP (data not shown). At that time, we suspected that VSW-3 RNAP might not function to activate the pVSW-3 promoter in E. coli.

2.2 The hint from the genome

The sequence of the untranslated region (UTR) upstream of the coding sequence of the gene of interest (GOI), including promoter and ribosome binding site (RBS), plays a critical role in transcription initiation in bacteria (7). To determine whether the sequence of pVSW-3 was sufficient for VSW-3 RNAP, we found all the sequence between the last open reading frame (ORF) and the start codon of the structure gene on the genome VSW-3 phage then cloned this sequence (BBa_K4907043) upstream the reporter gene gfp to construct a reporting circuit (BBa_K4907122) containing the promoter and RBS of VSW-3 phage. Surprisingly, E. coli BL21(DE3) carrying this reporting circuit and VSW-3 RNAP-harboring plasmid exhibited mild green fluorescence after supplemented with the inducer L-arabinose (Fig. 1). This meant that the VSW-3 RNAP might function well to activate the UTR sequence from the genome and implied the reason why the original report circuit failed to function might result from the 18-bp pVSW-3 promoter sequence itself.

Then we turned to check the original sequence we used. Aligned with the suspected promoter (UTR) on the genome of VSW-3 phage, we found that the promoter sequence from genome carried "GTA" at the 3' end (Fig. 2a). In addition, the researchers also explored the initiation efficiency of different nucleotides for the synthesis of transcripts in vitro and found that the nucleotide "G" downstream the promoter sequence might be necessary, which was similar to the T7 RNAP (Fig. 2b) (5). Based on what we observed and the previous work, we decided to adjust the 3' end sequence of the promoter pVSW-3 to see whether the change would result in a functional one in vivo or not.

2.3 pVSW-3(18) and pVSW-3(GGG)

By adding three redundant nucleotides, GTA or GGG, downstream the pVSW-3 respectively, we created two additional promoters, pVSW-3(18) (BBa_K4907012) and pVSW-3(GGG) (BBa_K4907015). Then the corresponding reporting circuits were constructed (BBa_K4907109 and BBa_K4907112) and characterized as well. After being induced by arabinose at 25 °C for 12 h, the group of pVSW-3(18) promoter showed a relatively stronger output signal than the other groups (Fig. 3), which indicated that the extra nucleotides GTA did contribute to the functional promoter sequence. Although the output signals of both pVSW-3(GGG) promoter and pVSW-3(genome) promoter were not as strong as the pVSW-3(18) promoter, they could also be distinguished from the control group (BBa_I0500) which contained no gfp in the bacteria. In summary, the practice of adding extra nucleotides downstream of the sequence of defect pVSW-3 promoter has generated functional promoters with the existence of VSW-3 RNAP and further characterizations were more focused on the pVSW-3(18) promoter.

3. VSW-3 expression system: basal expression and orthogonality to T7 system

Considering that there is a certain homology between VSW-3 RNAP and T7 RNAP (5), to improve the reliability of our experimental results in E. coli BL21(DE3), the leakage expression of pVSW-3(18) promoter without the existence of VSW-3 RNAP (or basal expression) should be determined and the potential expression caused by the endogenous T7 RNAP of BL21(DE3) need to be investigated as well.

3.1 Leakage of pVSW-3(18)

The reporting circuit of pVSW-3(18) (BBa_K4907108) was co-transformed with the control plasmid (BBa_I0500_pSB1C3) and VSW-3 RNAP-harboring plasmid (BBa_K4907114_pSB1C3) into BL21(DE3), respectively. Actually, the VSW-3 RNAP group here acted as the positive control, and the group containing no gfp was set as the negative control (“Control” for convenience). After induced by arabinose, the normalized fluorescence intensity of the group which has no VSW-3 RNAP expressed showed no significance with that of the Control (Fig. 4), which indicated the leakage expression of pVSW-3(18) was the same as the background of BL21(DE3), in another word, nearly no leakage expression of pVSW-3(18) promoter could be detected. The promising result also implied that the housekeeping RNA polymerase of E. coli<i> cannot recognize and activate the pVSW-3(18) promoter, which was similar to the T7 system as reported (8), resulting in the very stringent control that would further contribute to the construction of AND gate and even semantic containment for the biosafety concerns (9).

Fig. 4 The basal expression of VSW-3 system was characterized at 25 °C. <I>p</i>-value: no significance (ns), 0.0021 (**).

3.2 Orthogonality between VSW-3 and T7 system

Since the T7 system was widely used for synthetic biology and the certain homology between VSW-3 RNAP and T7 RNAP (even pVSW-3(18) and pT7), we were curious about the potential interaction(s) between the two systems. Due to the existence of chromosome-integrated T7 RNAP that could be induced by isopropyl <i>β-D-thiogalactoside (IPTG) in BL21(DE3) (or other strains with λ-DE3 integration), the reporting circuits of different promoters (BBa_K4907108 for pVSW-3(18) and BBa_K4907107 for pT7) could be transformed into the bacteria without cloning the coding sequence (CDS) of T7 RNAP into a second vector. After inducing the expression of T7 RNAP in BL21(DE3), the group of pT7 showed the strongest output signals, and no significant difference between the pVSW-3(18) and the control group (pET-28a(+) inserting no genes) could be observed (Fig. 5a), which indicated that only the T7 promoter could be activated by the T7 RNAP. For the tests of VSW-3 RNAP, the reporting circuits were co-transformed with the VSW-3 RNAP-harboring plasmid used before into E. coli DH10β which cannot metabolize L-arabinose due to the araD139 mutation. By contrast, only the pVSW-3(18) could be activated by the VSW-3 RNAP after arabinose induction rather than pT7 (Fig. 5b). Based on these observations, it was convinced that the VSW-3 system is orthogonal to the T7 system (Fig. 5c), which indeed enriched the available orthogonal parts library for the applications of synthetic biology.

In summary, the VSW-3 expression system has nearly no leakage in E. coli, and the good orthogonality between the VSW-3 and T7 system can be further applied to the construction of a multi-input AND gate for gene expression and even a novel expression strain.

4. Optimal temperature

4.1 The optimal temperature of VSW-3 system

Since the low-temperature inducible expression, or a cold-responsive expression pattern, was the need for achieving the goal of anti-icing, the optimal temperature of the VSW-3 system should be investigated as well. The bacteria (BL21(DE3)) harboring VSW-3 RNAP and the reporting circuits of pVSW-3(18) was cultivated at different temperatures after induction respectively. Among the temperatures we tested, the VSW-3 system has the strongest activity at 25 °C (at least 4-fold higher than other groups) (Fig. 6a), which was consistent with the results in vitro (5). Besides, the VSW-3 RNAP functioned better at 15 °C than 30 °C and physiological 37 °C, which implied the obvious low-temperature preference of this RNA polymerase. Due to the limited time and the number of available thermostats in our lab, more detailed temperature gradients need to be examined and we hoped that this could be achieved in the future.

4.2 The tests of T7 system

Similar tests were carried out for the T7 system by using the BL21(DE3) harboring the reporting circuit of pT7 (BBa_K4907107), focused on the relatively lower temperatures we were interested in (15, 25, 30 °C). As expected, with the increase in temperature, the T7 system showed an increasing trend of activity (Fig. 6b), which exhibited an obviously different temperature preference compared to the VSW-3 system. It should be noted that the inducing system used to trigger the expression of RNA polymerase and the position of the CDS of RNA polymerase were totally different between the two systems, hence we were hard to assert that the T7 system has higher efficiency than the VSW-3 system at 25 °C and more conditions should be rigorously controlled if the comparison is expected to be meaningful.

5. The attempts to improve efficiency

Although the VSW-3 system functioned well at 25 °C with the pVSW-3(18) as the output promoter, however, the efficiency of this system still should be improved if the application scope of the novel system is expected to be broadened. Here, we have come up with two approaches for improving the system’s efficiency from the aspect of VSW-3 RNAP and the cognate promoter, respectively.

5.1 The positive feedback circuit

Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis (10, 11). Leveraging the design of positive feedback circuits, the gene amplifiers could be constructed and has been widely implemented in the field of whole-cell biosensor (12-19). Based on the intention of creating a positive feedback circuit to improve the efficiency of VSW-3 system, placing the amplifier protein (here, VSW-3 RNAP) under the control of the amplifier promoter (here, pVSW-3(18)) and the responsive promoter (here, BBa_I0500) would result in the pattern of positive feedback (Fig. 7a). Specifically, the bicistronic design, BBa_K4907113 was constructed on the backbone pSB3K3. As performed in BL21(DE3) like before, the normalized fluorescence intensity of each group was calculated after induced by arabinose for 12 hours. However, no amplification effects of this bicistronic design were observed when compared to the non-positive-feedback one (Fig. 7b), despite the fact that the output signal of the positive feedback circuit was much higher than that of the control (BBa_I0500) as well. The unsatisfied result might be attributed to the metabolic burden due to the relatively long sequence of the new reporting circuit with the whole CDS of VSW-3 RNAP added downstream or the possibly low efficiency of VSW-3 RNAP to transcribe the long DNA sequence. Setting the CDS of VSW-3 RNAP under the control of its cognate promoter pVSW-3(18) as an independent transcriptional unit may alleviate the issues. We hoped that this would be further investigated in the future due to the time limit of this competition season.

< center>Fig. 7 The attempt to construct a positive feedback circuit. a Graphic description of the positive feedback design. b Characterizations for testing the amplification effect at 25 °C in BL21(DE3).</center> Anyway, this attempt of constructing a positive feedback circuit represented the one aspect for improving the efficiency. In addition, increasing the dosage of the CDS of VSW-3 RNAP or reporter gene would be another available choice for amplification (20).

5.2 The pVSW-3 series

Inspired by the test for different lengths of promoters in vitro (5), we wondered whether changing the length of pVSW-3(18) would improve the efficiency of VSW-3 system. Hence, the pVSW-3(16), pVSW-3(17), and pVSW-3(19), which were only distinguished by the number of “T” nucleotide upstream (Fig. 8a), were chosen to construct a reporting circuit on pSB3K3 backbone, respectively (BBa_K4907108, BBa_K4907110 and BBa_K4907130). The efficiency of pVSW-3 series promoters was determined as characterizations performed in BL21(DE3) like before and the output signals were all normalized to the pVSW-3(18) promoter. As shown in Fig. 8b, the pVSW-3(19) exhibited a stronger output signal than the previously created pVSW-3(18), which indicated that VSW-3 RNAP functioned better with this pVSW-3(19) promoter rather than other promoters. As for pVSW-3(16) which gained about 60% activity of the original pVSW-3(18) promoter, we might regard this weaker one as a better choice for the certain context demanding lower transcriptional strength (21).

In short, this attempt to change the promoter length represented the other aspect of improving the VSW-3 system’s efficiency. Both the changes of VSW-3 RNAP and the promoter pVSW-3(18) would contribute to the changes in the efficiency of the VSW-3 system, whether from the aspect of detailed sequences or the dosage of specific parts. Massive variants can be generated by many mutation means and leveraging various emerging high-throughput screening technologies will further develop and generate more efficient VSW-3 systems (21).

6. The construction of AND gate

6.1 Introduction

Since we chose the cspA system as the cold-responsive parts and constructed CspA CREC (please see BBa_K4907118 for more information) for low-temperature induced expression of target protein for our anti-icing project, however, obvious leakage expression of this system at high temperatures was observed. For the sake of reducing the leakage of the CspA CREC system at high temperatures (such as 37 °C), we have come up with the approach of building an AND-logic gate for tightly controlling the expression of CspA CREC. One idea was based on the classic hrp system (22), and the other, here, was based on the split-intein (23) combined with the novel VSW-3 system. In our design, the VSW-3 RNAP was split into two halves and fused to the split intein SspC and NpuN respectively. And each fusion half was placed under the control of cspA promoter (pCspA). In this time, the two pCspA promoters acted as inputs while the pVSW-3(18) promoter played the role of output with the target genes placed downstream. Theoretically, leakage expression will occur at a certain probability for a single pCspA as output, however, when the pVSW-3(18) is set as the output, the leakage at high temperatures will rarely happen due to the low-temperature preference of VSW-3 RNAP even if the leakage of two pCspA promoters occur.

6.3 Regaining function due to the intein

We then turned to investigate the function of split intein and if the junction would result in an intact form of the polymerase with regaining normal function. The characterization circuit (BBa_K4907117) was constructed on the backbone pSB1C3 by placing the SspC-VSW-3 RNAPC and VSW-3 RNAPN-NpuN under the control of L-arabinose induced promoter BBa_I0500 in a bicistronic pattern. We first explored the expression of these variants in the host BL21(DE3). The bacterial culture that was cultivated at 25 °C with inducer or not was made the sample for subsequent SDS-PAGE analysis. For the groups with inducer added, the target bands of the split VSW-3 RNAP, VSW-3 RNAPN-NpuN (66.1 kDa), and SspC-VSW-3 RNAPC (40.3 kDa), could be observed around 66 kDa and between 35 and 42 kDa, respectively. While, strong bands between 80 kDa and 95 kDa could also be observed in the lanes of the group with inducer added, which was consistent with the size of intact VSW-3 RNAP (90.6 kDa) that could be clearly seen in the lanes of the VSW-3 RNAP-expressing positive control group (left of the marker, with inducer added of course) (Fig. 11a). This indicated that the split intein could function well at 25 °C to self-splice then generate the intact form of VSW-3 RNAP. Then, the reporting circuit of pVSW-3(18) was co-transformed with BBa_K4907117_pSB1C3 into BL21(DE3) for testing the function of the resulting intact VSW-3 RNAP generated by the junction of the split intein. After induction at 25 °C for 20 hours, the group expressing the split halves of VSW-3 RNAP exhibited a much stronger output signal than that of the control group (BBa_I0500_pSB1C3) (Fig. 11b), proving that the generated intact VSW-3 RNAP also gained its normal function. Despite this, the output signal of this experimental group was not as strong as the native intact one, which might be attributed to the decreasing junction efficiency of the split intein at 25 °C.

These results indeed laid the foundation for constructing transcriptional AND gate as our design. It was unprecedented that the novel RNA polymerase split into two fragments with the split intein SspC and NpuN in our chosen site could form the intact one due to the junction of intein and regain its normal function to activate the cognate pVSW-3(18) promoter. Besides, since the orthogonality between the T7 system and the VSW-3 system, the split of the VSW-3 RNAP would also be used in parallel for more complex network engineering (27).

6.4 Combining CspA CREC and VSW-3 RNAP

The efficiency of VSW-3 RNAP when loaded into the CspA CREC system (please see more information in BBa_K4907118) should be determined, since we planned to achieve a design with AND-logic gate to reduce pCspA leakage. Therefore, we constructed the pCspA-B0034-VSW-3 RNAP-B0015 circuit (CspA CREC-VSW-3 RNAP) on the backbone pSB1C3 (BBa_K4907119_pSB1C3) and co-transformed this plasmid with the reporting circuit of pVSW-3(18) (BBa_K4907109_pSB3K3) into E. coli BL21(DE3). After induction at 20 °C for 20 hours, even the temperature might not be the optimal temperature for VSW-3 RNAP, the output signal of the RNA polymerase loaded into CspA CREC was much stronger than that of the control group without harboring VSW-3 RNAP (Fig. 12). Hence, it was convinced that the combination of VSW-3 RNAP with CspA CREC could function well. It was very promising to achieve the goal of constructing a cold-responsive transcriptional AND gate.

6.5 The multi-input AND gate

For the final goal of constructing AND-logic gate based on the split VSW-3 RNAP to reduce the leakage of cspA-mRNA expression system, we have endeavored verifying every key point involved in the AND gate. Due to the low-temperature preference of VSW-3 RNAP, it was convinced that the low-temperature (25 °C in this test) would be set as an intrinsic input of the multi-input AND-logic gate. However, we met some challenges when cloning VSW-3 RNAPN-NpuN into the CspA CREC system, so we decided to clone this sequence into the classic cold-inducible vector (28). pCold I, in which the cold-responsive mechanism is also based on the function of cspA-mRNA that just distinguishes from the CspA CREC in few sequences. At first, we tried to co-transform the pCold I-VSW-3 RNAPN-NpuN (No part name specified with partinfo tag._pCold I), L-arabinose-induced SspC-VSW-3 RNAPC expression circuit (BBa_K4907116_pSB1C3) and the reporting circuit of the pVSW-3(18) promoter (BBa_K4907109_pSB3K3) into BL21(DE3). It should be noted that the cspA promoter on pCold I is an IPTG-inducible one because a copy of lacO is placed downstream the promoter sequence. Hence, in accordance with the ways of induction, the AND gate constructed here was a three-input one, linking the chemogenetics and thermogenetics (Fig. 13a, left). We set carefully the conditions of different control groups and measured the output signals after induction for 12 hours. As expected, when all the induction requirements were met (0.5 mM IPTG, 0.2% L-arabinose (m/v), cultivated at 25 °C), the normalized fluorescence intensity was significantly strongest (Fig. 13a, right). Besides, when one input was absent (“0”), the output signals were even about 4-fold lower than the “all-input-1” group and all these deficient groups exhibited an equal level of the weak output signals, which indicated that the three-input AND gate was very stringent.

Fig. 13 TBD Then, the L-arabinose-induced SspC-VSW-3 RNAPC expression circuit (BBa_K4907116_pSB1C3) was replaced by CspA CREC-SspC-VSW-3 RNAPC (BBa_K4907121_pSB1C3), in which the split half of RNA polymerase is under the control of cspA-mRNA system that needs no inducers at all. Therefore, a two-input AND gate was formed (Fig. 13b, left). Similar experiment was performed as mentioned above while the input of arabinose was removed, and the results demonstrated that the pVSW-3(18) promoter could be activated only when both genes of split halves were induced by IPTG and the low-temperature (Fig. 13b, right). Given the conditions that IPTG was added, only the relative low cultivating temperature (25 °C) would result in the “1” state of output. In other word, the strategy of leveraging the tight control of AND-logic gate to reduce the leakage expression of CspA CREC system was sufficiently feasible. In summary, combining CspA CREC and the split intein VSW-3 RNA polymerase can generate a modular and orthogonal genetic multi-input AND-logic gate like the hrp system reported before (29). What’s more, we believed that by replacing the input promoters and introducing another logic gates into it, the applications of the split intein VSW-3 RNA polymerase will be further developed and expanded.

7. The construction of neo expression vectors and engineered bacteria

T7 RNAP is a polymerase from T7 phage, and the expression system composed of it with PT7 has a high efficiency. It has been widely used in the field of protein expression and purification in E. coli and even has a specially constructed specific bacteria such as E. coli BL21(DE3) and its corresponding specific expression vector pET series plasmid. However, high efficiency and by-products of the transcription process have also become the problems facing its further utilization. VSW-3 RNAP and T7 RNAP are homologous to a certain extent, and even the corresponding promoter sequences of the two are only 8 bp different in the middle. Through the characterization of the above parts, we found that VSW-3 RNAP, as a new phage RNA polymerase, has a wider application value in E. coli. Its low-temperature activity makes it possible for it to have a unique effect on the expression of proteins. Therefore, we decided to construct a specific expression bacteria and corresponding expression vector, so that this polymerase can play a role in more occasions.

7.1 The birth of pLOVE

In order to further improve the low-temperature specificity of the VSW-3 expression system, pLOVE plasmid was created based on the structure of the cspA expression system. lacO and cspA 5’-UTR are attached after pVSW-3(18), and cspA 3’-UTR is attached after CDS. In addition, the polyclonal sites of pET series vectors were added between the two UTR sequences, which can improve the suitability of pLOVE. When users have the need to replace CDS sequences, they only need to use the original enzyme restriction sites that comply with the rules of T7 expression vectors. Fig. x is the experimental result of the expression of green fluorescent protein by pLOVE, and Fig. x is the result of the expression of xxx protein by pLOVE. The results showed that pLOVE was able to work properly in E. coli. 缺实验结果

7.2 Beyond BL21(DE3)

After constructing the corresponding expression vector, our further work is to integrate VSW-3 RNAP into the genome of E. coli, but the current work has not been completed.

8. Conclusion（可以再宏大一些）

We found an RNA polymerase, VSW-3 RNAP, which has good activity at low temperatures and completed its characterization in E. coli. Not only does it fulfill the original purpose of our design, but we also found that it has special value in the field of low-temperature expression. On the one hand, we explored various promoters that might match the VSW-3 RNAP, and in a series of attempts to improve its efficiency, we found the most efficient promoter, pVSW-3(19). On the other hand, we constructed its specific expression vector pLOVE and attempted to integrate VSW-3 RNAP into the genome of E. coli. Our aim is to obtain a specific expression bacteria and its corresponding expression vector. This means that our work can create an entirely new bacteria of cryophilic clones with unique low-temperature specificity and protein expression advantages. It has even more value for future iGEMers and researchers, and it may be useful in unexpected places. In terms of logic gate, in addition to the VSW-3 RNAPN-NpuN and SspC-VSW-3 RNAP themselves can be controlled by different input signals, we found that temperature also has a certain degree of rigor in inducing VSW-3 RNAP, so on the basis of our original design, we introduced a logic gate with signal input to achieve three logics and gates, which can cope with some special situations. In xxx (school), three-input control (blue light, red light, double light) was achieved using blue light and red light promoters, but the leakage of optogenetics was relatively serious. In this case, the three-logic and gate based on VSW-3 RNAP and gate can play a more important role.

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Sequence and Features