Part:BBa_K819005

Constitutive LuxBrick Generator

a fusion protein consisting of E.coli endogenous SOS system repressor LexA and fungus N.crass photosensor protein VVD with LexA carrying mutation at position 40-42 and VVD carrying mutation N56K, C71V and M135I. This part serves as an ultra sensitive photoreceptor(which can sense light as weak as moonlight) and will induce a light-dependent repression of genes with their promoter containing mutated 408 form of SOS box.

Luminesensor is designed by following the general principle of optogenetics fusion protein design--attaching a physiologically functional domain to a photoreceptor domain.

FIG.1 The general design of optogenetic fusion protein.

In order to circumvent the potential problems of current optogenetic approaches, we concluded that our Luminesensor should be highly sensitive, should incorporate no chromophore that cannot be synthesized by bacteria, and should be modular in structure and function. We gladly found that among all these three groups of commonly used light-sensitive domains, phototropins possesses the most distinct modular structure. Phototropins have a structurally conserved light sensor domain, termed LOV (light, oxygen and voltage) domain, which is easily discernable and often precedes, within a single reading frame, a sequence of an enzymatically functional domain, connected by the sequence of a linker domain. So far our good phototropin has satisfied two of our requirements: modularity and compatibility. So how about its sensitivity? To date, scientists have, for example, attached Rac1 protein to LOV2 domain from phototropin1 originated from Avena sativa to achieve photoactivatable cell motility, or shuffled the histidine kinase domain of FixL protein, which belongs to a two-component system, to the downstream of the LOV domain of B. subtilis YtvA protein to create a light-regulated histidine kinase, and so on. Among these designs, a particular one that utilizes the photosensor domain of a Vivid (VVD) protein, which originated from Neurospora. Crassa, camptured our attention. The VVD-GAL fusion protein thus designed achieved a rather high light-sensitivity—about 0.04W/m2. This is definitely thrilling news. Moreover, VVD was shown to form a rapidly exchanging homodimer upon blue-light activation, which means, like the Phy-PIF system, the general theme of protein-protein interaction might also be applied to this particular protein. Adding more promise to this VVD protein is its size—the smallest LOV domain containing protein known. This feature makes it easy to engineer and more likely to be stably expressed in bacteria systems.

FIG.2 Illustration of function mechanism of phototropin VVD.

Based on the discussion above, we have finally chosen the smallest LOV protein (or phototropin) VVD protein’s photosensor domain as our photoreceptor domain. Due to its small size, structural and functional modularity, bacteria compatibility, previous experience of being highly sensitive and its photoswitching mechanism, we reasoned that VVD photosensor domain would be an enabling tool in our coming design of a novel optogenetic module.

Our next step is to choose a physiologically functional domain for our new optogenetic module.

Based on the function mechanism of phototropin VVD, we can envisage a rough blueprint of our design: the DNA binding domain of a transcription inhibitor shall be fused to the downstream of VVD photosensor domain, and upon blue-light activation, VVD domain will dimerize, helping the DNA binding domain dimerize at the same time to enable its DNA binding activity and inhibition of transcription initiation. Based on these criteria, a transcription repressor,LexA, came into our sight. LexA is a transcription repressor of all the genes in the SOS system in E.coli, and its crystal structure has been resolved at high-resolution. LexA protein consists of a N-terminal DNA binding domain and a C-terminal dimerization domain with a short hydrophilic linker linking the two separate domains, making the structure impressively modular. Under normal physiological conditions, two LexA proteins will form a homodimer through the dimerization domain interface and bind to the SOS box target gene sequence in promoters of genes in SOS system and create significant steric hindrance for transcription polymerase binding and thus inhibit transcription initiation.

FIG.3 Illustration of function mechanism of LexA transcription repressor.

From these facts, we can conclude that the LexA repressor is the suitable physiological functional domain for our light-sensitive module, as it matches all of our criteria. First of all, a high-resolution crystal structure clearly shows its structural modularity. Beside the apparent structural modularity, various researches have utilized LexA to create versatile gene expression regulating modules that have convincingly demonstrated its functional modularity, further adding to its promise. (For example, a LexA-based genetic system has been devised for monitoring and analyzing protein heterodimerization in Escherichia coli.) Moreover, the DNA binding domain of LexA rigorously requires dimerization to bind efficiently to its targeting site (SOS box), and DNA binding domain itself has no dimerization ability. Last but not least, LexA is bacteria endogenous protein, which means it is likely to be efficiently expressed and function normally in bacteria systems.

FIG.4 Illustration of the function mechanism of our luminesensor.

By choosing the DNA binding domain of a bacteria endogenous transcription repressor, we must use reporter genes that will also be repressed by endogenous LexA protein. If we do not introduce changes into the DNA binding domain of our luminesensor, the only way to rule out the interference of endogenous LexA would be knocking out the genomic sequence of LexA protein. This will indubitably diminish its applicability in the future, since it will not work in any strain with endogenous LexA remaining. So we set out to find a solution to this important problem. The most natural way would be creating a mutated form of LexA so that it will only recognize a target sequence different from the sequence the wild-type LexA would recognize. We gladly found that such a thing actually exists. It is called the LexA408 variant, which carries mutations PA40, NS41 and AS42 and has been shown much higher affinity for a symmetrically altered target sequence CCGT (N)8 ACGG, different from the wild type SOS box CTGT (N)8 ACAG, which is specifically recognized by wild-type LexA. Thus by introducing the 408 mutations into the DNA binding domain of our luminesensor, we can insulate our light-sensitive system from the interference from the genetic context of the host strain. This outstanding character that have ensured the orthogonality of our system to the endogenous SOS system will greatly enhance the compatibility of our module to those synthetic modules that have hitherto been designed and thus add to the promise of future application.

FIG.5 Our solution to the problem of orthogonality.

In order to rationally optimize our Luminesensor’s dynamic range and decay half-life, we need to build a dynamic model in the first place to simulate our Luminesensor system and determine which ones of those parameters in the system would be decisive to the two dynamic features we are interested in.

We listed all possible states of the molecules involved in our Luminesensor system and transitions between those states and created an ordinary deferential equation system based on law of mass action to describe the dynamic behavior of our system. After in-silico simulation, we singled out 4 critical parameters in our system that would determine the above two aspects of our system’s dynamic performance.

FIG.6 Kinetic Network of our Luminesensor.

Two of them will affect the background-signal ratio, namely the VVD protein dimerization equilibrium constant and dimerized Luminesensor’s DNA binding equilibrium constant.

Having singled out these four critical parameters, we set out to search through literature to find mutations that would enhance them. We found that there does not seem to exist mutations that would enhance dimerized Luminesensor’s DNA binding equilibrium constant. Rather, those mutations seem to undermine the DNA binding affinity, which would be detrimental to our system. We further reasoned that any mutation that would facilitate the disassociation of monomer Luminesensors from target DNA sequence is also likely to undermine the DNA binding affinity. So we focused on looking for VVD photosensor domain mutations that would promote VVD dimerization and VVD decay. We eventually localized on two specific mutations of VVD photosensor domain: a M135I mutation that will enhance the VVD dimerization, and a I74V mutation in the vicinity of FAD molecule that will enhance the VVD decay rate.

Having determined the plausible mutations, when then introduced mutation M135I, I74V and the combination of the two mutations into our 408 form of Luminesensor. By characterizing the dynamic behavior of the mutated Luminesensor along time course, we discovered that mutation M135I indeed improved the signal-background ratio significantly. However, I74V mutation seems to reduce the signal-background ratio dramatically, rendering our Luminesensor useless. So disregarding of the faster decay half-life it may provide, we have to rule I74V out of our design.

Sensitivity

We tested the sensitivity of Luminesensor by examine the light-dependent transcriptional activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitated the observation of transcriptional activity. Based on the consideration of guaranteeing accuracy and precision, our setup (Figure 7):consists of three central parts: light source, incubator and 48-well plate.

FIG.7 The set-up of sensitivity tests.

On account of high sensitivity, protecting the system from the preventable light exposure with the purpose of acquiring the accurate results which is the true reflection of our sensitivity is necessary. In order to solve the problems, we focus on two foremost aspects: utilizing attenuators to weaken the light intensity and using tin foil to avoid light leakage. For more details about how we conducting the experiment, see experiment procedures. In our experiments, illumination with different light intensity conditions at 460nm peak light from blue LED arrays for 16 hours show marked light-depressed reporter gene transcription, which indicates that under different blue light exposure conditions, there was hardly any light-induced reporter gene transcriptional activity. But when in the dark environment (packaged with three layers of aluminum foil), our systems showed extremely high GFP expression (Figure 8):

FIG.8 The sensitivity of Luminesensor.

Orthogonality Test

It is our biggest concern whether lexA408 Luminesensor works independently of endogenous LexA. If yes, maximizing its biological orthogonality will make Luminesensor a really plug-and-play device.

Two sections of testing expriments were carried out simultaneously. GFP was selected as a reporter and was fused downstream to 2 promoters (psulA408 and precA408) controlled by Luminesensor with 408 mutation and to 2 promoters (psulA and precA) controlled by Luminesensor without mutation. GFP expression is expected to have a negative relation with repression activity. To be more specific, higher level of green fluorescent indicates weaker repression effect; lower expression of GFP stands for stronger repression.

1. Experimental Design To prove the orthogonality, facts that LexA408-VVD and endogenous LexA work totally independently are needed. Considering practical efficiency, 2 points of evidence are to collect: 1. Promoters psulA and precA are repressed in wild-type Ecoli strains while promoters psulA408 and precA408 are not blocked in wild-type strains; 2. LexA408-VVD Luminesensor efficiently represses its target under blue illumination, while it does not repress targets in total dark.

FIG.9 The design of orthogonality tests.

Below is a collage of our plates. The plates are placed in groups at 30℃ in either total dark or blue illumination. Visual results fit well with Figure 1.Visual look of plates were positive evidence for us.

FIG.10 Plate streaked with wild type E.coli transformed with wild type luminesensor and GFP fused to LexA responsive promoters, and cultivated in the dark (A), or under light (B).

FIG.11 Plate streaked with wild type E.coli transformed with GFP gene fused to the downstream of psulA408 (A) and precA408 (B)..

FIG.12 Plate streaked with wild type E.coli transformed with mutated luminesensor and GFP gene fused to the downstream of precA408 and psulA408 and cultivated in the dark or under light.(A: luminesensor + precA408-GFP, cultivated in the dark. B: luminesensor + precA408-GFP, cultivated under light. C: luminesensor + psulA408-GFP, cultivated in the dark. D: luminesensor + psulA408-GFP, cultivated under light.)

Improving Dynamic Performance

To test whether our designated mutations would improve the dynamic performance of Luminesensor in respect to reversibility and the on/off ratio, we co-transformed the psulA-GFP (GFP driven by luminesensor repressible promoter psulA) plasmid with four versions of Luminesensor plasmid into BL21 (ΔLexA ΔSulA): the original LexA-VVD(WT), LexA-VVD(I74V), LexA-VVD(M135I), and LexA-VVD(I74V+M135I). Resulted four strains were designated as LV-WT, LV-74, LV-135, LV-74-135, respectively. The overnight culture of the four strains were diluted 500 times and divided into two groups: one exposed to blue light and the completely wrapped with aluminum foil. After incubation for 16 hours, GFP expression levels were measured. As shown in figure below, LV-135 has an increased on/off ratio compared to the original LexA-VVD, while the LV-74 and LV-74-135 show reduced on/off ratios, which is in accordance with our modeling result.

FIG.13 Effects of introduced mutations on the performance (on/off ratio) of Luminesensor.

In order to demonstrate the reversibility of our Luminesensor, we recorded the different target gene expression in different dark time using RFP as the reporter. The expression of RFP increases with the increase of dark time.

FIG.13 The time course of RFP expression.

Reference
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Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 3014
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2012
  Illegal XhoI site found at 2842
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4401
  Illegal BsaI.rc site found at 1410
  Illegal SapI.rc site found at 4726