From c04abe55c28d1f5cdb656e16bc483fafb2e64558 Mon Sep 17 00:00:00 2001
From: Hao Li <2798778230@qq.com>
Date: Tue, 5 Dec 2023 12:17:05 +0000
Subject: [PATCH] Update file results.html
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</p>
</div>
- <!--5-Exploration of the expression system of VSW-3 --->
- <div class="myPage-paragraph">
- <h5 class="myPage-paragraph-headline-h5"><b>Exploration of the expression system of VSW-3 </b></h5>
- <h6 class="myPage-paragraph-headline-h6"><b>pVSW-3(18) could form an efficient expression system with VSW-3
- RNAP </b></h6>
- <p class="myPage-paragraph-content">
- The sequence of untranslated region (UTR) upstream the coding sequence of gene of interest (GOI), including promoter and ribosome binding site (RBS), plays a critical role in transcription initiation in bacteria. 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 (<a href=" http://parts.igem.org/Part:BBa_K4907043"
-style="text-decoration:none;">BBa_K4907043</a>) upstream the reporter gene <i>gfp</i> to construct a reporting circuit (<a href=" http://parts.igem.org/Part:BBa_K4907022"
- style="text-decoration:none;">BBa_K4907122</a>) containing the promoter and RBS of VSW-3 phage.
- </p>
-
- <p class="myPage-paragraph-content">
- Surprisingly, <i>E. coli</i> BL21(DE3) carrying this reporting circuit and VSW-3 RNAP-harboring plasmid
- exhibited mild green fluorescence after supplemented with the inducer <i>L</i>-arabinose (Fig. 14). 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.
- </p>
-
- <!---Fig. 14--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/14.png" alt="">
- <div class="myPage-paragraph-fig-description"><b>Fig. 14 The image of bacterial culture upon blue light
- illumination.</b> Left: no <i>gfp</i> contained. Right: the test of UTR sequence with VSW-3 RNAP
- expressed.
- </div>
- </div>
-
- <p class="myPage-paragraph-content">
- 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.
- 15a). In addition, the researchers also explored the initiation efficiency of different nucleotides for the
- synthesis of transcripts <i>in vitro</i> and found that the nucleotide "G" downstream the promoter sequence
- might be necessary, which was similar to the T7 RNAP (Fig. 15b). 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 <i>in vivo</i> or not.
- </p>
-
- <!---Fig. 15--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/15.png" alt="" style="width:70%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 15 The hint from genome. a</b> Alignment of the
- sequence from VSW-3 genome and the original pVSW-3. <b>b</b>Comparing the initiation efficiency of
- different initiation nucleotides for in vitro transcription in the previous work.
- </div>
- </div>
-
- </div>
-
- <!--6 -pVSW-3(18) and pVSW-3(GGG) --->
- <div class="myPage-paragraph">
- <h6 class="myPage-paragraph-headline-h6"><b>pVSW-3(18) and pVSW-3(GGG)</b> </h6>
- <p class="myPage-paragraph-content">
- By adding three redundant nucleotides, GTA or GGG, downstream the pVSW-3 respectively, we created two
- additional promoters, pVSW-3(18) (<a href=" http://parts.igem.org/Part:BBa_K4907012"
- style="text-decoration:none;">BBa_K4907012</a>) and pVSW-3(GGG) (<a
- href=" http://parts.igem.org/Part:BBa_K4907015" style="text-decoration:none;">BBa_K4907015</a>). Then
- the corresponding reporting circuits were constructed (<a href=" http://parts.igem.org/Part:BBa_K4907109"
- style="text-decoration:none;">BBa_K4907109</a> and
- <a href=" http://parts.igem.org/Part:BBa_K4907112" style="text-decoration:none;">BBa_K4907112</a>) and
- characterized as well. After induced by arabinose at 25 °C for 12 h, the group of pVSW-3(18) promoter showed
- a relative stronger output signal than the other groups (Fig. 16), 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 that strong as the pVSW-3(18) promoter, they
- could also be distinguished from the control group (<a href=" http://parts.igem.org/Part:BBa_I0500"
- style="text-decoration:none;">BBa_I0500</a>) which
- contained no <i>gfp</i> in the bacteria. In summary, the practice of <b>adding extra nucleotides downstream
- the sequence of defect pVSW-3 promoter has generated functional promoters</b> with the existence of VSW-3
- RNAP and further characterizations were more focused on the pVSW-3(18) promoter.
- </p>
- <!---Fig. 16--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/16.png" alt="">
- <div class="myPage-paragraph-fig-description"><b>Fig. 16 Characterization of pVSW-3(18), pVSW-3(GGG) and
- pVSW-3(genome) promoters at 25 °C.</b> <i>p</i>-value: 0.0021 (**), 0.0002 (***), <0.0001 (****).
- </div>
- </div>
- </div>
-
- <!--5 -The VSW-3 expression system is low leakage and orthogonal to the T7 expression system --->
- <div class="myPage-paragraph">
- <h5 class="myPage-paragraph-headline-h5"><b>The VSW-3 expression system is low leakage and orthogonal to the T7
- expression system</b> </h5>
- <p class="myPage-paragraph-content">
- Considering that there is a certain homology between VSW-3 RNAP and T7 RNAP, to improve the reliability
- of our experimental results in <i>E. coli</i> 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.
- </p>
-
- </div>
-
- <!--6-Leakage of pVSW-3(18) --->
- <div class="myPage-paragraph">
- <h6 class="myPage-paragraph-headline-h6"><b>Leakage of pVSW-3(18)</b> </h6>
- <p class="myPage-paragraph-content">
- The reporting circuit of pVSW-3(18) (<a href=" http://parts.igem.org/Part:BBa_K4907108"
- style="text-decoration:none;">BBa_K4907108</a>) was co-transformed
- with the control plasmid (BBa_I0500_pSB1C3) and VSW-3 RNAP-harboring plasmid (<a href=" http://parts.igem.org/Part:BBa_K4907114"
- style="text-decoration:none;">BBa_K4907114</a>_pSB1C3) into
- BL21(DE3), respectively. Actually, the VSW-3 RNAP group here was acted as the positive control and the group
- containing no <i>gfp</i> 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 was showed
- no significance with that of the Control (Fig. 17), which indicated the leakage expression of pVSW-3(18) was
- as the same as the background of BL21(DE3), in other word, <b>nearly no leakage expression of pVSW-3(18)
- promoter could be detected</b>. The promising result also implied that the housekeeping RNA polymerase of
- <i>E. coli</i> cannot recognize and activate the pVSW-3(18) promoter, which was similar to the T7 system as
- reported, resulting in the very stringent control that would further contribute to the construction of
- AND gate and even semantic containment for the biosafety concerns.
- </p>
-
- <!---Fig. 17--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/17.png" alt="" style="width:25%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 17 The basal expression of VSW-3 system was
- characterized at 25 °C.</b> <i>p</i>-value: no significance (ns), 0.0021 (**).
- </div>
- </div>
-
- </div>
-
- <!--6-Orthogonality between VSW-3 and T7 system --->
- <div class="myPage-paragraph">
- <h6 class="myPage-paragraph-headline-h6"><i>Orthogonality between VSW-3 and T7 system </i></h6>
- <p class="myPage-paragraph-content">
- 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 so 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</i>-D-thiogalactoside (IPTG) in BL21(DE3) (or other strains with <i>λ</i>-DE3 integration), the reporting circuits of different promoters (<a href=" http://parts.igem.org/Part:BBa_K4907108"
- style="text-decoration:none;">BBa_K4907108</a> for pVSW-3(18) and <a href=" http://parts.igem.org/Part:BBa_K4907107"
- style="text-decoration:none;">BBa_K4907107</a> 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. 18a), which indicated that only the T7 promoter could be activated by the T7 RNAP. While for the tests of VSW-3 RNAP, the reporting circuits were co-transformed with the VSW-3 RNAP-harboring plasmid used before into <i>E. coli</i> DH10β which cannot metabolize L-arabinose due to the <i>araD139</i> mutation. By contrast, only the pVSW-3(18) could be activated by the VSW-3 RNAP after arabinose induction rather than pT7 (Fig. 18b). Based on these observations, <b>it was convinced that the VSW-3 system is orthogonal to the T7 system</b> (Fig. 18c), which indeed enriched the available orthogonal parts library for the applications of synthetic biology.
- </p>
-
- <!---Fig. 18--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/18.png" alt="" style="width:70%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 18 Orthogonality between T7 system and VSW-3 system.
- a</b> Characterizations in BL21(DE3) for testing the function of T7 RNAP to different reporting circuits
- (pET-28a(+) as Control) at 25 °C. <b>b</b> Characterizations in DH10β for testing the function of VSW-3
- RNAP to different reporting circuits (BBa_I0500 as Control) at 25 °C. <b>c</b> Graphic description of the
- orthogonality between the two systems. <i>p</i>-value: no significance (ns), <0.0001 (****).
- </div>
- </div>
-
- <p class="myPage-paragraph-content">
- In summary, the VSW-3 expression system has nearly no leakage in <i>E. coli</i>, and the well orthogonality
- between the VSW-3 and T7 system can be further applied to the construction of multi-input AND gate for gene
- expression and even a novel expression strain.
- </p>
-
- </div>
-
- <!--5-The VSW-3 system showed excellent low temperature induction effect --->
- <div class="myPage-paragraph">
- <h5 class="myPage-paragraph-headline-h5"><b>The VSW-3 system showed excellent low temperature induction
- effect</b> </h5>
- <h6 class="myPage-paragraph-headline-h6"><b>The optimal temperature of VSW-3 system</b> </h6>
- <p class="myPage-paragraph-content">
- 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. 19a), which was
- consistent with the results <i>in vitro</i>. Besides, the VSW-3 RNAP functioned better at 15 °C than 30
- °C and physiological 37 °C, which implied the obvious <b>low-temperature preference</b> of this RNA
- polymerase. Due to the limit of 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.
- </p>
-
- <!---Fig. 19--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/19.png" alt="" style="width:80%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 19 Characterizations of the temperature effect for
- VSW-3 system (a) and T7 system (b).</b></div>
- </div>
-
- </div>
-
- <!--6-The tests of T7 system --->
- <div class="myPage-paragraph">
- <h6 class="myPage-paragraph-headline-h6"><b>The tests of T7 system</b> </h6>
- <p class="myPage-paragraph-content">
- The similar tests were carried out for T7 system by using the BL21(DE3) harboring the reporting circuit of pT7 (<a href=" http://parts.igem.org/Part:BBa_K4907107"
- style="text-decoration:none;">BBa_K4907107</a>), focused on the relative lower temperatures we were interested in (15, 25, 30 °C). As expected, with the increase of temperature, the T7 system showed an increasing trend of activity (Fig. 19b), 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.
- </p>
-
- </div>
-
- <!--5-pVSW-3(19) and pVSW-3(17) can improve the efficiency of the VSW-3 expression system --->
- <div class="myPage-paragraph">
- <h5 class="myPage-paragraph-headline-h5"><b>pVSW-3(19) and pVSW-3(17) can improve the efficiency of the VSW-3
- expression system</b> </h5>
- <p class="myPage-paragraph-content">
- 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.
- </p>
- </div>
-
- <!--6-The positive feedback circuit --->
- <div class="myPage-paragraph">
- <h6 class="myPage-paragraph-headline-h6"><b>The positive feedback circuit</b> </h6>
- <p class="myPage-paragraph-content">
- 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. 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. Based on the intension of creating a positive feedback circuit to improve the
- efficiency of VSW-3 system, <b>placing the amplifier protein (here, VSW-3 RNAP) under the control of the
- amplifier promoter (here, pVSW-3(18)) and the responsive promoter (here, <a
- href=" http://parts.igem.org/Part:BBa_I0500" style="text-decoration:none;">BBa_I0500</a>) would result
- in the pattern of positive feedback (Fig. 20a).</b> Specifically, the bicistronic design, <a
- href=" http://parts.igem.org/Part:BBa_K4907113" style="text-decoration:none;">BBa_K4907113</a> 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. 20b),
- despite the fact that the output signal of the positive feedback circuit was much higher than that of the
- control (<a href=" http://parts.igem.org/Part:BBa_I0500" style="text-decoration:none;">BBa_I0500</a>) as
- well. While, the unsatisfied result might be attributed to the metabolic burden due to the relative 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.
- </p>
-
- <!---Fig. 20--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/20.png" alt="" style="width:80%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 20 The attempt of constructing a positive feedback
- circuit. a</b> Graphic description of the positive feedback design. <b>b</b> Characterizations for testing
- the amplification effect at 25 °C in BL21(DE3).
- </div>
- </div>
-
- <p class="myPage-paragraph-content">
- Anyway, <b>this attempt of constructing a positive feedback circuit represented the one aspect for improving
- the efficiency</b>. In addition, increasing the dosage of the CDS of VSW-3 RNAP or reporter gene would be
- another available choice for amplification.
- </p>
-
- </div>
-
<!--6-The pVSW-3 series --->
<div class="myPage-paragraph">
- <h6 class="myPage-paragraph-headline-h6"><b>The pVSW-3 series</b> </h6>
- <p class="myPage-paragraph-content">
- Inspired by the test for different lengths of promoters in vitro, 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. 21a), were chosen
- to construct reporting circuit on pSB3K3 backbone, respectively (<a
- href=" http://parts.igem.org/Part:BBa_K4907108" style="text-decoration:none;">BBa_K4907108</a>, <a
- href=" http://parts.igem.org/Part:BBa_K4907110" style="text-decoration:none;">BBa_K4907110</a> and <a
- href=" http://parts.igem.org/Part:BBa_K4907130" style="text-decoration:none;">BBa_K4907130</a>). The
- efficiency of pVSW-3 series promoters were 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. 21b, the
- pVSW-3(19) exhibited a stronger output signal than the previous 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.
- </p>
-
- <!---Fig. 21--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/21.png" alt="" style="width:80%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 21 The attempt of changing the promoter length. a</b>
- Alignment of pVSW-3 series promoters. <b>b</b> Characterizations for testing the efficiency of different
- promoters at 25 °C in BL21(DE3). <i>p</i>-value: no significance (ns), 0.0332 (*), <0.0001 (****).
- </div>
- </div>
-
- <p class="myPage-paragraph-content">
- In short, <b>this attempt of changing the promoter length represented the other aspect for improving VSW-3
- system's efficiency</b>. Both the changes of VSW-3 RNAP and the promoter pVSW-3(18) would contribute to the
- changes of efficiency of VSW-3 system, whatever from the aspect of detailed sequences or the dosage of
- specific parts. Massive variants can be generated by many mutations means and leveraging various emerging
- high throughput screening technologies will further develop and generate more efficient VSW-3 systems.
- </p>
-
- </div>
-
- <!--4-The full-length polymerase was split into two polypeptides to construct the AND-logic gate --->
- <div class="myPage-paragraph">
- <h4 class="myPage-paragraph-headline-h4"><b>The full-length polymerase was split into two polypeptides to
- construct the AND-logic gate</b> </h4>
- <p class="myPage-paragraph-content">
- Since we chose the <i>cspA</i> system as the cold-responsive parts and constructed CspA CREC (please see <a
- href=" http://parts.igem.org/Part:BBa_K4907118" style="text-decoration:none;">BBa_K4907118</a> 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 CspA CREC system at high temperatures (such as 37 °C), we have come up with the approach of
- building AND-logic gate for tightly controlling the expression of CspA CREC. The one idea was based on the
- classic <i>hrp</i> system (*) , and the other, here, was based on the split-intein (*) 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 <i>cspA</i> promoter
- (pCspA). In this time, the two pCspA promoters acted as inputs while the pVSW-3(18) promoter played the role
- as output with the target genes placed downstream. Theoretically, leakage expression will occur at a certain
- probability for a single pCspA as output, however, <b>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</b> even if the
- leakage of two pCspA promoters occur.
- </p>
-
- <!---Fig. 22--->
- <div class="myPage-paragraph-fig">
- <img src="https://static.igem.wiki/teams/4907/wiki/result/result/22.png" alt="" style="width:80%;">
- <div class="myPage-paragraph-fig-description"><b>Fig. 22 Structure prediction.</b><b> a</b> The predicted
- structure of VSW-3 RNAP. The split site was colored in blue. <b>b</b> The predicted structure of VSW-3
- RNAPN-NpuN (<a href="http://parts.igem.org/Part:BBa_K4907118"
- style="text-decoration:none;">BBa_K4907018</a>). The NpuN was colored in light pink.
- <b>c</b> The predicted structure of SspC-VSW-3 RNAPC (<a href=" http://parts.igem.org/Part:BBa_K4907118"
- style="text-decoration:none;">BBa_K4907107</a>).
- The SspC was colored in light pink.
- </div>
- </div>
-
- <p class="myPage-paragraph-content">
- Therefore, the most important issue was the split site of VSW-3 RNAP. The split intein SspC and NpuN was
- reported to construct a split T7 RNA polymerase and build a transcriptional AND-logic gate(*). For keeping
- the self-splicing activity of the intein, according to the previous experience, the N-terminal splice
- junction is tolerant of noncanonical sequences(*), the +1 Cys residue in the C-extein serves as a
- nucleophile during the splicing process and is therefore essential(*). Because we did not want to introduce
- any mutation into the VSW-3 RNAP, we looked for any natural occurrence of “CFN†in the VSW-3 RNAP sequence.
- <b>As the exact sequence does not exist, we decided to split the VSW-3 RNAP between amino acids 477 and
- 478</b>, yielding “AW†as the N-terminal extein junction sequence and “CFE†as the C-terminal extein
- junction sequence. The structure of whole-length and split forms of VSW-3 RNAP were predicted <i>via</i> the
- server of <a href=" https :// colab . research . google . com / gi thub / deepmind / alphafold / blob / main / notebooks / AlphaFold . ipynb" style="text-decoration:none;">AlphaFold2</a>, with the chosen residues forming part of an α-helix that were colored in blue (Fig.
- 22).
- </p>
-
- <p class="myPage-paragraph-content">
- Modular verifications were implemented step by step in this section. We firstly confirmed the function of
- split intein and then combined the VSW-3 RNAP with CspA CREC, finally, constructed a three-input AND-logic
- gate.
- </p>
-
- </div>
+
<!--5-The functional defect of split VSW-3 RNAP --->
<div class="myPage-paragraph">
@@ -835,46 +505,6 @@ style="text-decoration:none;">BBa_K4907043</a>) upstream the reporter gene <i>gf
</div>
<!--5-mv140-linker-cbm-his and mv140-linker-his --->
- <div class="myPage-paragraph">
- <h5 class="myPage-paragraph-headline-h5"><b>The multi-input AND gate</b></h5>
- <p class="myPage-paragraph-content">
- For the final goal of constructing AND-logic gate based on the split VSW-3 RNAP to reduce the leakage of <i>cspA</i>-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 <i>cspA</i>-mRNA that just distinguishes from the CspA CREC in few sequences.
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- At first, we tried to co-transform the pCold I-VSW-3 RNAPN-NpuN (<partinfo>BBa_ K4907148</partinfo>_pCold I), <i>L</i>-arabinose-induced SspC-VSW-3 RNAPC expression circuit (<partinfo>BBa_K4907116</partinfo>_pSB1C3) and the reporting circuit of the pVSW-3(18) promoter (<partinfo>BBa_K4907109</partinfo>_pSB3K3) into BL21(DE3). It should be noted that the <i>cspA</i> promoter on pCold I is an IPTG-inducible one because a copy of <i>lacO</i> 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% <i>L</i>-arabinose (m/v), cultivated at 25 °C), the normalized fluorescence intensity was significantly strongest (Fig. 13a, right). Besides, when one input was absent, 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.
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- Then, the <i>L</i>-arabinose-induced SspC-VSW-3 RNAPC expression circuit (<partinfo>BBa_K4907116</partinfo>_pSB1C3) was replaced by CspA CREC-SspC-VSW-3 RNAPC (<partinfo>BBa_K4907121</partinfo>_pSB1C3), in which the split half of RNA polymerase is under the control of <i>cspA</i>-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). <b>Given the conditions that IPTG was added, only the relative low cultivating temperature (25 °C) would result in the “1†state of output</b>. 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.
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- 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 <i>hrp</i> 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.
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- <h5 class="myPage-paragraph-headline-h5"><b>The temperature specificity of the VSW-3 expression system further improves the effect of reducing leakage at high temperatures</b> </h5>
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- Further, based on the work in the previous few sections, we found that because the VSW-3 RNAP itself has a strong temperature specificity, this can be used as an input signal to help us construct a three-input and gate. Therefore, we used <a href=" http://parts.igem.org/Part:BBa_K4907116" style="text-decoration:none;">BBa_K4907116</a>_pSB1C3 (C-terminal polypeptide), pCold-VSW-3 RNAPN-NpuN and <a href=" http://parts.igem.org/Part:BBa_K4907108" style="text-decoration:none;">BBa_K4907108</a>_pSB3K3 (pVSW-3(18) reporting circuit) realizes three input signals, Ara, IPTG and temperature.
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- As shown in Fig. 26, when all three input signals are input normally, the normalized fluorescence intensity is significantly stronger than that of other experimental groups, and the results show that the three-input logic and gate designed based on VSW-3 RNAP can work normally.
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- <div class="myPage-paragraph-fig-description"><b>Fig. 26 Fluorescence intensity under three different input conditions</b>
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