Part:BBa_K598000

TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015

This is a GFP generator regulated by thiamine pyrophosphate (TPP)-responsive ribozyme 2.5. Upon adding TPP, the expression level of downsteam GFP would be down-regulated, and the extent of down-regulation corresponds to different concentrations of TPP.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 121
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 794
  

Background

Hammerhead ribozymes are small self-cleaving RNAs, first discovered in satellite RNAs of plant viruses that catalyze a specific phosphodiester bond isomerization reaction in the course of rolling-circle replication [1]. More recently a full-length hammerhead ribozyme from Schistosoma mansoni is being more frequently utilized for application. As shown in Fig.1A, this hammerhead ribozyme can be truncated to a minimal, catalytically active motif consisting of three base-pairing stems (marked in colors) flanking a central core of 15 mostly invariant nucleotides (marked in frame). And the conserved central bases are essential for the hammerhead ribozyme’s catalytic activity [1]. The tertiary structure shown in Fig.1B indicates that the secondary structure of the Schistosoma hammerhead ribozyme can be distorted into a uridine turn because of distant loop/bulge interaction which induces changes in stem II while simultaneously unwinding stem I. For the basic catalytic function of hammerhead ribozyme, the active site for self-cleaving of Schistosoma hammerhead ribozyme resides between stem III and stem I, as shown in Fig.1A.

It has been reported previously that mRNAs encoding enzymes involved in thiamine (vitamin B1) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the assistance from protein cofactors [2]. These ligand-binding mRNAs actually possess thiamine or pyrophosphate binding domain, called aptamer, in which the binding event can bring about a conformational change which is important for genetic control. This natural thiamine pyrophosphate (TPP) aptamer can bind to TPP specifically and a defined structure is stabilized. As shown in Fig.1C, upon addition of TPP, TPP can bind loop in green through non-covalent bond. Fig.1D shows the tertiary structure of natural TPP aptamer binding to TPP.

Figure 1 The schematic structures of Schistosoma hammerhead ribozyme and natural TPP aptamer. A) The secondary structure of Schistosoma hammerhead ribozyme. Three base-pairing stems are shaded in colors. The part in yellow represents stem III, which is later modified to be the linker between hammerhead ribozyme and aptamer. The part in blue represents stem II, and the purple and red ones stand for two parts of stem I. The sequence in frame represents for the conserved nucleotides. The red arrow points to the scissile bond. B) The tertiary structure of Schistosoma hammerhead ribozyme. The cyan part indicates the fragment of mRNA after cleavage. The red one indicates the active site for self-cleavage. The linker between hammerhead ribozyme and aptamer is shown in yellow. C) The secondary structure of natural TPP aptamer. TPP can bind to loop in green through non-covalent bond and the part marked in yellow indicates the linker between hammerhead ribozyme and apatamer. D) The tertiary structure of natural TPP aptamer. The three-dimensional segment in blue is TPP, and the yellow part represents the linker between Schistosoma hammerhead ribozyme and natural TPP aptamer. Nucleotides that bind to TPP are shown in green.

Original Design of TPP Ligand Responsive Hammerhead Ribozyme

In fact, the natural aptamer domain of the TPP riboswitch can be exploited to construct very efficient ribozyme-based artificial switches that regulate gene expression, demonstrated by Markus Wieland et al.[3]. To couple the natural TPP aptamer riboswitch with Schistosoma hammerhead ribozyme, stem III of Schistosoma hammerhead ribozyme and yellow shaded stem of TPP aptamer in Fig.1C were modified to construct linker between hammerhead ribozyme and aptamer. The resulting artificial ribozymes functioned with high performance, whose highest fold reached 1000.

Markus Wieland et al. have created several mutants of the constructed ribozyme-based TPP-responsive artificial ribozyme switches (TPP ribozyme)[3]. We chose two of the mutants in our project, one of which can activate downstream gene expression upon adding TPP, numbered 1.20, and the other would inhibit downstream gene expression when TPP added, numbered 2.5. The secondary structure of TPP ribozyme 2.5 is shown in Fig.2. The lower part of the structure is natural TPP aptamer riboswitch, and the upper part is Schistosoma hammerhead ribozyme. Stem III in green indicates the linker between aptamer and hammerhead ribozyme, the pairing nucleotides of which is the only distinction between TPP ribozyme 1.20 and 2.5.

Figure 2 Secondary structure of artificial thiamine pyrophosphate (TPP) ribozymes 2.5. Natural TPP aptamer domain (blue) is fused to stem III of the Schistosoma hammerhead ribozyme. The linker between aptamer and hammerhead ribozyme is shown in green. Stems are indicated by roman numerals; rate-enhancing interaction between stem I and stem II are shown as gray lines; the cleavage site is marked by a red arrow. RBS is shaded in pink and the translation start code (AUG) is shaded in black. The figure was modified from [3].

Figure 3 Allosteric mechanism of TPP ribozymes. Upon self-cleavage the RBS would be released from pairing, thus ribosome could get access to RBS and initiate translation of the downstream gene .Upper) Mechanism for TPP ribozyme 1.20. Upon addition of TPP, TPP ribozyme 1.20 adopts a conformation that facilitates the self-cleavage of hammerhead domain. Lower) Mechanism for TPP ribozyme 2.5. When TPP added, TPP ribozyme 2.5 would change to a conformation that hinders the self-cleavage of hammerhead domain. Red: RBS sequence, blue: natural TPP aptamer, green: linker between aptamer and hammerhead domain, black: Schistosoma hammerhead ribozyme, red arrow: self-cleavage site. The figure is modified from [3].

The ribosomal binding site (RBS) of TPP ribozyme locates at the extended stem (shaded red in Fig.2). The Schistosoma hammerhead domain in TPP ribozyme could perform self-cleavage when posed in an appropriate conformation, and upon self-cleavage the RBS would be released from pairing, thus ribosome could get access to RBS and initiate translation of the downstream gene. Though similar in secondary structure, TPP ribozyme 1.20 and 2.5 undergo different mechanisms to regulate the translation of downstream gene. Upon addition of TPP, the aptamer domain would bind to TPP; while TPP ribozyme 1.20 would change to a conformation that is suitable for hammerhead domain to cleave itself, TPP ribozyme 2.5 would undergo a conformational change that would decelerate the self-cleaving rate of hammerhead domain. Therefore, upon adding TPP, TPP ribozyme 1.20 would facilitate the translation of downstream gene, whereas TPP ribozyme 2.5 would decrease the translation strength of downstream gene (Fig.3).

Manipulating and Experimental Data

We designed several experiments to demonstrate that TPP ribozymes are truly modular RNA controllers, independent of sequence context. Firstly we constructed Part BBa_K598000(Fig.4). This part consists of TPP ribozyme 2.5 kindly provided by Prof. Hartig’s lab, BBa_E0040 and BBa_B0015.

Figure 4 Construction of TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015. This part consists of TPP ribozyme 2.5 with native RBS (AAGGAGAT), BBa_E0040 and BBa_B0015. It is obtained by PCR from inactive TPP-HHAz 2.5 [3] which is kindly provided by Markus Wieland et al., and then inserted into pSB1C3 through standard assembly.

Then we constructed three different constructs based on this part, along with one construct kindly provided by Prof. Hartig’s lab (Fig.5). The plasmid provided by Prof. Hartig’s lab contain TPP ribozyme 2.5 with an upstream T7 promoter, and the downstream coding sequence is a GFP gene, followed by a T7 terminator (Fig.5A). We termed this construct T7-2.5 (BBa_K598016).

To prove that the performance of TPP ribozymes is not relevant to the downstream coding sequence, we inserted first 36 base pairs of the coding sequence from Part BBa_E0040 ahead of the GFP coding sequence into the plasmid (Fig. 5B). The construct was termed 36-2.5.

To further investigate whether the performance of TPP ribozymes is influenced by upstream promoter and downsteam terminator, we inserted pBAD promoter (BBa_I13453) into the upstream of BBa_K598000 and constructed BBa_K598011(Fig.5C). This construct was named pBAD-2.5.

Another plasmid were constructed by inserting first 36 base pairs of CI gene ahead of GFP coding sequence in pBAD-2.5 (Fig.5D). This was named CI-2.5.

Figure 5 Scheme of constructs designed to demonstrate the modularity of TPP ribozymes. A) T7-2.5 (BBa_K598016)consists of T7 promoter, TPP ribozyme, GFP and T7 terminator with native RBS. B) 36-2.5 were constructed by inserting the first 36 base pairs of BBa_E0040 between TPP ribozyme and GFP. C) pBAD-2.5 (BBa_K598011)consists of pBAD promoter (BBa_I13453) , TPP ribozyme, (BBa_E0040) and (BBa_B0015) with native RBS (AAGGAGAT). D) CI-1.20/CI-2.5 were constructed by adding the first 36 base pairs of CI ahead of coding sequence of E0040.

After transforming these four constructs, namely T7-2.5, 36-2.5, pBAD-2.5, and CI-2.5, into E. coli DH5α cells respectively, the bacteria were characterized in M9 medium with TPP concentration gradient. The result turned out that corresponding working curves of the four constructs overlapped to a large extent (Fig.6), despite of different downstream coding sequence (comparing T7-2.5 to 36-2.5, or pBAD-2.5 to CI-2.5), or different upstream promoter or downstream terminator (comparing T7-2.5 to pBAD-2.5). These experiments conclusively demonstrated that the performance of TPP ribozyme 2.5 was independent of their coding sequence context, rendering it a truly modular RNA device to regulate gene expression.

Figure 6 Working curves of TPP ribozyme 2.5 in different constructs. The inhibition ratio is fluorescence intensity under given TPP concentrations compared to that of without TPP. Constructed plasmids were transformed into E. coli DH5α cells and characterized in M9 medium with a TPP concentration gradient of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 uM. T7-2.5 and 36-2.5 were induced by 1mM IPTG. pBAD-2.5 and CI-2.5 were induced by 1mM arabinose.

Applications

This year our team developed a platform for soft-coding of genetic circuits aiming at making screening fast, affordable and more predictable. The platform is composed of a RNA controller toolkit and an RBS calculator as illustrated in our project. To demonstrate the versatility and validity of the platform, we utilized the platform to improve performance of two modular genetic devices, AND gate and bistable switch. TPP hammerhead ribozyme performs well during characterization, thus is elected as an component of the toolkit to optimizing these devices.

In order to optimize the performance of AND gate, we firstly determined the optimal translation strength of the T7ptag gene using our RNA controller toolkit. By placing TPP hammerhead ribozyme 2.5 upstream of the coding sequence, we obtained an AND gate modulator whose T7ptag gene translation rate varies in response to TPP concentration (Figure 7a). By optimizing the strength of translation, we are able to make up for the leakage in transcription and a translation rate that endows the AND gate with satisfactory performance (Figure 7b).

Figure 7 Optimization of AND gate performance using RNA controller(TPP ribozyme).(a) Output fluorescence of the AND gate device without addition of TPP ligand(corresponding to a △G of -5.78kJ/mol). (b)Output fluorescence of the AND gate device with addition of maximal concentration of TPP ligand(1μM，corresponding to a △G of -3.38kJ/mol). Vertical and horizontal axes indicate logarithm of the concentrations of arabinose and salicylate respectively. Apparently, addition of TPP ligand(which attenuates translation strength) improved the AND gate performance by decreasing the area of region for “ON” state. The two output color plots are mapped to their corresponding positions in the full phase diagram in Figure 2, showing that they display fair agreement with modeling results(white and yellow rectangular respectively).

TPP 2.5 is also introduced into the bistable switch part (BBa_K228003) that inherited from Peking 2007 iGEM team.(For the full information of bistable switch, please refer to BBa_K598002) A bistable switch with TPP 2.5 modifying the translation rate of cI434 gene is constructed. (BBa_K598024) (Figure 8)

Figure 8 Construction of the bistable switch device carrying the RNA controller (TPP ribozyme, shown as the tuning switch named TPP2.5 in the figure).

We set two experiment groups for characterizing this part: one without addition of TPP and another with TPP sufficient for full induction of the RNA controller’s functions (self-cleavage of ribozyme). The experimental results are shown in Figure 9. It can be seen that the group with excess TPP (down-regulated translation strength of cI434 gene) indeed displayed bistability. Thus, it has been indicated that TPP ligand responsive hammerhead ribozyme will be suitable for modifying nonlinear and non-Boolean logic genetic device like bistble switch.

Figure 9 Fluorescence stereomicroscopic images of E.coli colonies with and without TPP treatment.(A)E.coli colonies without TPP treatment(no decrease in translation rate) are all green(high CI434/low CI state), displaying monostability of the genetic device. (B)E.coli colonies with TPP treatment(no decrease in translation rate) are a mixture of green(high CI434/low CI state) and red(low CI434/high CI state) colonies, displaying bistability of the genetic device. (C)Experimental results are mapped to the simulated “green” proportion–△G curve.

We further applied this platform to optimize a segment of violacein biosynthetic pathway, and achieved producing purer desired products. To see more information, please go to BBa_K598019.

Figure 10 (A) Design of constructs to fine-tune violacein biosynthetic pathway. Hexagon: Stem-loop terminator (BBa_B0015); Bent arrow: pBAD promoter ((BBa_I13453); Oval: Ribosomal binding site; Straight arrow: Coding sequence originated from Part:BBa_K274003; ribbon shape: TPP-responsive ribozyme. (B) Decreasing the amount of VioE may lower the pool of prodeoxyviolacein, hence lowering the production of deoxychromoviridans, thus we inserted a TPP down-regulated hammerhead ribozyme (TPP ribozyme 2.5) in front of vioE, and the metabolic flux would favor proviolacein. Dash arrow: down-regulated. (C) Increasing the amount of VioD may increase the yield of proviolacein, thus we inserted a TPP up-regulated hammerhead ribozyme (TPP ribozyme 1.20) upstream of vioD, and the metabolic flux would favor proviolacein. Dash arrow: down-regulated; Filled arrow: up-regulated.

Figure 11 HPLC results of the bacterial extraction when different concentrations of TPP were presented in the cultures. When induced with arabinose, the bacterial extraction produced two peaks eluted at 0.7-1.0 min ((b) comparing to (a)), and we tentatively assumed that the two peaks corresponded to proviolacein (left) and deoxychromoviridans (right). Upon adding TPP into the cultures, the amount of deoxychromoviridans decreased, and the ratio between proviolacein and deoxychromoviridans increased upon increasing TPP concentration ((b) to (f)), until deoxychromoviridans was not detectable (f).

Supporting Information

Additionally, to further confirm that the rise or drop of the working curves in Fig.6 on different TPP concentrations was indeed the contribution of TPP ribozyme 2.5, we constructed a plasmid as control by substituting TPP ribozyme 2.5 in pBAD-2.5 BBa_K598011 to the native RBS (AAGGAGAT) of TPP ribozyme 2.5, termed "TPP-RBS" (Fig.7). Another construct termed "pBAD-1.20" was also constructed by substituting TPP ribozyme 2.5 to TPP ribozyme 1.20 in pBAD-2.5 construct. Similar characterization was performed, and the result showed that the fluorescence intensity produced by TPP-RBS fluctuated, yet not significant enough to show a trend to increase or decrease when TPP concentration went up, compared with the obvious fluorescence intensity change produced by pBAD-2.5 or pBAD-1.20(Fig.8). Therefore, we can reach the conclusion that TPP ribozyme 2.5 functioned modularly to regulate downstream gene’s translation strength upon different concentrations of TPP.

Figure 7 Construction of TPP-RBS. TPP-RBS consists of pBAD promoter, native RBS, BBa_E0040 and BBa_B0015.

Figure 8 The fluorescence intensity of TPP-RBS, pBAD-1.20 and pBAD-2.5 under different TPP concentrations. Ordinate axis indicates the fluorescence intensity normalized by cell density. Constructed plasmids were transformed into E. coli DH5a cells and characterized in M9 medium with a TPP concentration gradient of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3uM, with induction by 1mM arabinose.

References

[1] Monika Martick and William G.Scott. (2006). Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis. Cell 126, 309-320

[2] Wade Winkler, Ali Nahvi Ronald R. Breaker. (2002). Thiamine Derivatives Bind Messenger RNAs Directly to Regulate Bacterial Gene Expression. Nature 419, 952-956

[3] Markus Wieland, Armin Benz, Benedikt Klauser, and Jörg S. Hartig. (2009). Artificial Ribozyme Switches Containing Natural Riboswitch Aptamer Domains. Angew. Chem. 121, 2753-2756