Design

Affi-PmrB/A System

Overview

In order to detect Sp10 protein on the sperm surface representing sperm motility, we constructed the Affi-PmrB/A system by modifying the TCS receptor PmrB with an affibody capable of recognizing the antibody of Sp10. We first carried out structural prediction and molecular docking simulation on our modified protein to verify the feasibility of our design in advance. Then we introduced the Affi-PmrB/A system into E. coli to validate the effectiveness of our design by detecting the fluorescence signals expressed by downstream reporter genes.

PmrB/A system and Affibody

Originated from Salmonella, PmrB/A system is a two-component signal transduction system capable of sensing the iron ions in the environment through its receptor PmrB and transcription factor PmrA.[1] To be more specific, when extracellular iron ions bind to the iron(III)-binding motif on receptor PmrB, PmrB will phosphorylate the transcription factor PmrA, which then binds to promoter PmrC and initiates downstream gene transcription (Fig. 1A).
Originated from proteins in Staphylococcus aureus, affibody can specifically bind to the Fc peptide of an antibody. The simplified affibody consists of two α helixes, and its structure is similar to the ligand recognition region of receptor PmrB.[2] Such a coincidence inspired us to replace the iron(III)-binding motif on receptor PmrB with affibody so that the combined system can possess the potential of responding to the target protein signal.
Here in our design, the engineered E.coli can use the specific antibody as an intermediary to respond to Sp10 protein on the sperm surface. When Sp10 is detected, the signal can be relayed through the Affi-PmrB/A system and presented by fluorescence signals of downstream reporter genes (Fig1B).


Schematic diagram of PmrB/A system (A) and Affi-PmrB/A system(B).
Fig 1. Schematic diagram of PmrB/A system (A) and Affi-PmrB/A system(B).

How did we come up with this design?

Previous detection systems based on engineered bacteria mostly rely on transcription factors and specific promoters that bind to the substance to be tested, thus the sensors using engineered bacteria could only detect small molecules that can enter the cell. In order to detect the protein signal on the sperm surface, new bacterial-based protein detection system is needed.

In the NEU iGEM 2020 project, they developed a bacterial-based method to detect the S protein on the surface of coronavirus through PmrB/A system. NEU team replaced the iron (III)-binding motif of PmrB with the core domain of angiotensin converting enzyme 2 (ACE2) of human, successfully constructing a system to detect the SARS-CoV-2 S protein. Inspired by the high modifiability of affibody, we constructed the Affi-PmrB/A system to detect Sp10 protein on the sperm surface.

Parts assembly and characterization

We first designed the sequence of Affi-PmrB and performed structural prediction and molecular docking simulation to check the feasibility of our design. Following that, we introduced the Affi-PmrB system into E.coli for experimental verification. We designed two plasmids: pAffi-PmrB/A and pAff-PmrB/A-amplifier to improve fluorescence signal output.

pAffi-PmrB/A

Construction

Plasmid design of pAffi-PmrB/A.
Fig 2. Plasmid design of pAffi-PmrB/A.

  • The pET28a plasmid with kanamycin resistance is used as the backbone.
  • EGFP is placed downstream of the promoter PmrC as the reporter gene.
  • IPTG is used to induce the expression of receptor PmrB and transcription factor PmrA.
  • The human IgG antibody is used to induce the system to produce EGFP.
Characterization

To examine the efficiency of our design, we observed the fluorescence signal of EGFP under fluorescence microscope after induction. The expected result is that more intense fluorescence signal can be observed after IgG induction. We also applied fluorescence spectrophotometry to measure the fluorescence intensity.

pAffi-PmrB/A-amplifier

Construction

Plasmid design of pAffi-PmrB/A-amplifier.
Fig 3. Plasmid design of pAffi-PmrB/A-amplifier.

  • The pET28a plasmid with chlorampenicol resistance is used as the backbone.
  • EGFP is placed downstream of the promoter PmrC as the reporter gene.
  • IPTG is used to induce the expression of receptor PmrB and transcription factor PmrA.
  • The human IgG antibody is used to induce the system to produce EGFP.
  • T7-T3 cascade amplifier system is introduced into the genetic circuit. T7 core can only start transcription after binding withT3σ. The expression of T3σ is controlled by PmrC.
Characterization

To examine the efficiency of amplifier, we applied fluorescence spectrophotometry to measure the fluorescence intensity of EGFP signal by microplate reader. The expected result is that the fluorescence intensity of EGFP will be much higher with the amplifier introduced in the circuit.

Affi-NisK/R System

Overview

We introduced the nisin two-component system (TCS) of lactobacillus into E.coli, and reconstructed the inducer nisin into a fusion protein nisA-affibody containing nisin precursor nisA and affibody ZEGFR:2377 to recognize EGFR, which is a critical index of sperm fertility. The effectiveness of our modified sensing system was verified using different reporter systems. In addition, we experimentally validated the promoter strength results predicted by our software, obtaining multiple mutant promoters that can significantly enhance the strength of our TCS promoter.

NisK/R system

As a member of lantibiotics, nisin is produced by the lactic acid bacterium Lactococcus lactis and is characterized by a wide spectrum of antibacterial activity agasint gram-positive bacteria.[3] In Lactococcus lactis, nisin can induce its own synthesis through a two-component system——nisin TCS. The nisin TCS consists of the extracellular inducer nisin, the membrane-bound histidine kinase receptor nisK , the cytoplasmic response regulatory protein nisR, the promoter PnisA regulated by nisR and its downstream expression genes. After extracellular nisin binds to the receptor nisK, nisK can activate NisR through phosphorylation, and the activated NisR binds to promoter PnisA and induces the expression of nisin precursor peptide nisA.[4]


Schematic diagram of nisin TCS.
Fig 4. Schematic diagram of nisin TCS.

Nisin TCS can mediate cellular changes in respnse to environmental stimuli, thus we can modify it to accomplish our goal of using engineered bacteria to detect extracellular protein signals. Here in our design, nisA (precursor of nisin) is fused with the affibody ZEGFR:2377 to sense the existence of EGFR protein on the sperm surface indicating sperm fertility, and the expression gene downstream of the promoter PnisA is replaced with a reporter gene (EGFP or lacZ) to examine the effectiveness of our modified nisin TCS. When EGFR is detected, the signal can be relayed through the Affi-NisK/R system and presented by visualizable signals of downstream reporter genes.


Schematic diagram of our modified Affi-NisK/R system.

Fig 5. Schematic diagram of our modified Affi-NisK/R system.

How did we come up with this design?

After discovering the PmrB/A system as an excellent signal detection and transduction system, we kept searching for more two-component systems so that two TCSs can work together to recognize two different protein signals respectively. Through literature research, we cast our attention on nisin TCS in Lactococcus lactis because the function of each component was well-studied. Previous literatures have demonstrated the possibility of synthesizing nisin in bacteria. [5-7]Therefore, we decided to construct the fusion protein of nisA and EGFR antibody to achieve our goal of detecting EGFR on the sperm surface.

Parts assembly and characterization

Our wetlab verification of Affi-NisK/R system can be divided into three parts:

  1. Verification of overexpressed nisin TCS: pNisinOE1 and pNisinOE2 were constructed to validate that the overexpressed nisin TCS could function in E.coli.
  2. Verification of constituve nisinTCS: pConfocal was constructed to demonstrate the localization of NisK protein. pNisin1 and pNisin2 were constructed to create strains that can express NisK and NisR constitutively.
  3. Construction and verification of Affi-NisK/R system: pNisAB and pNisC were constructed to express NisA-affibody, which was then used as the inducer to induce pNisin2 to validate our Affi-NisK/R system.

pNisinOE1

Construction

Schematic diagram of pNisinOE1.
Fig 6. Schematic diagram of pNisinOE1.

  • The pET28a plasmid with kanamycin resistance is used as the backbone.
  • Receptor NisK and response transcription factor NisR are under the control of IPTG-inducible lac operator and T7 promoter.
  • The inducible promoter PnisA followed by EGFP as a reporter gene can be activated when nisin interacts with NisK and NisR.
Characterization

We cloned all the basic elements required in nisin TCS in pNisinOE1 to examine whether they can function correctly in E.coli. The characterization is divided into two parts:

  • Detect NisK and NisR expression by SDS-PAGE.
  • Measure EGFP fluorescence intensity by fluorescence spectrophotometry after nisin induction.

pNisinOE2

Construction

Schematic diagram of pNisinOE2.
Fig 7. Schematic diagram of pNisinOE2.

  • pNisinOE2 is derived from pNisinOE1.
  • The only difference is that the EGFP reporter gene is replaced with lacZα.
Characterization

Noticing that the EGFP fluorescence signal could only be observed under fluorescence microscope or microplate reader, we planned to use lacZ as the reporter gene to visualize its color reaction after adding X-gal.

pConfocal

Construction

Schematic diagram of pConfocal.
Fig 8. Schematic diagram of pConfocal.

  • The pFB20 plasmid (gift from Fang Ba) with chloramphenicol resistance is used as the backbone.
  • NisK and EGFP fusion gene are under the control of constitutive promoter J23100 and his operon terminator.
  • The inducible promoter PnisA followed by lacZα as a reporter gene. (unnecessary for localization observation)
Characterization

Since NisK is a membrane-bound receptor, we used pConfocal plasmid to confirm the localization of NisK under confocal laser scanning microscope.

pNisin1

Construction

Schematic diagram of pNisin1.
Fig 9. Schematic diagram of pNisin1.

  • The pFB20 plasmid (gift from Fang Ba) with chloramphenicol resistance is used as the backbone.
  • Receptor NisK and response transcription factor NisR are under the control of constitutive promoter J23100 and his operon terminator.
  • The inducible promoter PnisA followed by lacZα as a reporter gene can be activated when nisin interacts with NisK and NisR.
Characterization

To validate that NisK and NisR can be expressed constitutively, we observed the color reaction using pNisin1 after nisin induction to receive a preliminary qualitative result.

pNisin2

Construction

Schematic diagram of pNisin2.
Fig 10. Schematic diagram of pNisin2.

  • pNisin2 is derived from pNisin1.
  • The only difference is that the lacZα reporter gene is replaced with EGFP.
Characterization

To obtain quantitative results of constitutively expressing NisK and NisR, we applied fluorescence spectrophotometry and flow cytometry using pNisin2 after nisin induction.
To further improve the nisin TCS system, we designed a promoter strength prediction software to perform computer-based direction evolution of enhancing the strength of PnisA promoter. We intended to experimentally examine the promoter strength of the top10 mutant promoters with the highest predictive strength by fluorescence spectrophotometry and flow cytometry.

pNisAB & pNisC

Construction

Schematic diagram of pNisAB. Schematic diagram of pNisC.
Fig 11. Schematic diagram of pNisAB & pNisC.

  • The pET28a plasmid with kanamycin or chloramphenicol resistance is used as the backbone.
  • 6xHis tagged NisA-affibody, NisB, and NisC are under the control of IPTG-inducible lac operator and T7 promoter.
Characterization

pNisAB and pNisC were constructed to express the NisA-affibody fusion protein. The characterization is divided into two parts:

  • Purify NisA-affibody protein by Ni colomun and detect its expression by Western blots assay targeting 6xHis tag.
  • Measure EGFP fluorescence intensity by fluorescence spectrophotometry using our purified NisA-affibody to induce the constitutive nisin TCS.

Logic Gate

Overview

Our goal of the genetic circuit designed to characterize sperm quality is to examine sperm motility and fertility simultaneously. It is worth noting that these two indexes are correlated in a way that high motility is the premise of fertility. Therefore, we designed a three-state logic gate based on serine integrase Bxb1 and Cro/cI system to separate the two signals representing motility and fertility via conditional computation.
The aim of our logic gate system is to realize:
1) integration of different protein signals
2) logic computation of different signals
In addition, our logic gate system has the potential of a biological logic operator, given that all three basic logic gates (AND, OR, NOT gates) can be realized using serine integrase and Cro/cI system.


Skematic diagram of logic gate system in our genetic circuit design
Fig 12. Skematic diagram of logic gate system in our genetic circuit design.

Serine integrase

Serine integrase, adapted from bacteriophage, is capable of catalyzing site-specific recombination between two attachment sites, attP and attB. Depending on the relative location or orientation of recombination sites, three distinct recombination outcomes, integration, excision or inversion, can be realized.


Skematic diagram of three recombination outcomes catalyzed by integrase
Fig 13. Skematic diagram of three recombination outcomes catalyzed by integrase.

The advantage of serine integrase is that the recombination reaction driven by serine integrase is highly directional because it employs a double-strand break mechanism for recombination. Only in the presence of an accessory protein called a recombination directionality factor (RDF) can the reverse reaction take place. The ability to control reaction directionality has led to the development of serine integrase-based tools for controlled rearrangement and modification of DNA in synthetic biology, gene therapy, and biotechnology.[8]

Here in our design, a promoter is placed between two opposing attachment sites (attP and attB). Serine integrase Bbx1 is capable of inverting the DNA sequence flanked by these two attachment sites, thus changing the transcription direction. When integrase is not present, GFP will be expressed; in contrast, when integrase is present, RFP will be expressed.


Skematic diagram of how integrase functions in our logic gate design
Fig 14. Skematic diagram of how integrase functions in our logic gate design.

How did we come up with this design?

We first came across serine intergase in previous work [9], where they constructed a versatile method using three orthogonal serine integrases to manipulate different biobricks in vivo and in vitro. We realized that serine integrase could be employed to switch on the expression of different protein signals. Therefore, we introduced serine integrase into our design to achieve our goal of integrating different input signals and inducing corresponding output signal.

Cro/cI

Cro and cI participate in constituting a negative feedback system in bacteria phage λ, which triggers a binary switch that determines the fate of lysogenic bacteria. cI (also called λ repressor), can specifically associate with OR promoters and inhibit downstream gene expression. Cro is another repressor that blocks transcription of cI gene, thus derepressing the inhibition induced by cI. The function of Cro/cI system depends greatly on the special binding property of the OR regions, which is divided into three binding sites, namely OR1, OR2, OR3. cI binds more tightly to OR1 and OR2, and less tightly to OR3, while cro acts in an opposite way.


Skematic diagram of how cro/cI system works
Fig 15. Skematic diagram of how cro/cI system works.

Here in our design, serine integrase alone cannot accomplish our logic gate sytem because we cannot let a constitutive promoter keep expressing downstream reporter gene when no upstream signal is present. As a result, we introduced cro/cI system into our genetic circuit. To be more specific, the reporter gene (GFP or RFP) is placed downstream of the promoter OR1 and OR2, with which cI has the strongest binding affinity. In contrast, cI is placed downstream of the promoter OR3 because cro binds more tightly to OR3. In the case that no upstream signal is transduced to cro/cI system, cI would be expressed constitutively, repressing the expression of reporter gene GFP. However, as long as upstream signal exists, cro could be expressed to block cI transcription, thus derepressing the inhibition induced by cI and promoting reporter gene expression.


Skematic diagram of how cro/cI system functions in our logic gate design
Fig 16. Skematic diagram of how cro/cI system functions in our logic gate design.

How did we come up with this design?

In searching for genetic elements that could keep our reporter gene expression shut when no upstream signal is present, we came across the idea of applying operons to avoid undesired expression. However, operons were unsuitable in our system for two reasons. One reason was that we intended to detect protein signals rather than small molecules. Applying operons requires extra effort to include small molecules into our genetic circuit. Another reason was that small molecules could easily diffuse into the bacteria solution and cause interference.

Then we considered using antiterminators from bacteriophage to accomplish our goal, but abandoned this thought due to difficulties of detecing RNA process in our experiments. Finally, we came up with the idea of introducing cro/cI system from λ phage into our design. The reason it satisfied our demand is that cro/cI system exhibits a multi-level regulation and is well characterized by previous research. [10] Moreover, since cro/cI system is largely predesigned by nature, the incorporation of this system would not impose excessive burden on the host. In the lysogenic state of a wild type E.coli, the 256-AA (amino acid) lambda cI protein is only presented in 100-200 copies per cell[11], whereas the 66-AA Cro protein is present in less than 1,000 copies per cell[12]. Therefore, the burden of expressing these proteins in the host is expected to be minimal.

Logic gate design

Here we show our design in a global view. The overall genetic circuit consists of three levels of signal transduction:

  1. Surface protein detection: Protein signals detected from sperm surface is transform into ligands for engineered two-component system receptors using affibody.
  2. TCS (Two-component system) transduction: Engineered TCS could sense the ligands, relay the signals, and switch on specific promoters to transcribe cro or integrase.
  3. Logic gate operation: The two upstream signals are inetgrated in the logic gate system based on serine integrase and cro/cI. Different reporter genes will be expressed as the response to different upstream signals.

When Sp10, the index representing sperm motility, is recognized by the affibody attached to PmrB, it will trigger the downstream two-component system PmrB/A to express cro. Cro will then derepress the inhibition induced by cI, allowing downstream GFP expression. In contrast, when EGFR, the index representing sperm fertility, is recognized by affibody-nisin fusion protein, it will trigger the downstream two-component system NisK/R to express serine integrase Bxb1, which will invert the DNA sequence flanked by attP and attB sites to allow expression of mCherry. In this way, we realized our goal of transforming the sperm quality information into visualizable fluorescent protein signals.


Skematic diagram of our design
Fig 17. Skematic diagram of our design.

In addition, our logic gate system based on integrase and cro/cI is capable of realizing the three basic logic gates (AND, OR, and NOT gates). Consequently, our logic gate system could be regarded as a biological logic operator.


Skematic diagram of three basic logic gates (AND, OR, and NOT gates) achievde by integrase and cro/cI system
Fig 18. Skematic diagram of three basic logic gates (AND, OR, and NOT gates) achievde by integrase and cro/cI system. [13]

Parts assembly and characterization

In order to verify the function of each part in the logic gate system, we designed two plasmids: pLogic1 for integrase characteriztaion, and pLogic2 for cro/cI characterization.

pLogic1 for integrase characterization

Construction

Plasmid design of pLogic1 for inergase characterization
Fig 19. Plasmid design of pLogic1 for inergase characterization.

  • pBAD/araC mimics the TCS promoter NisK, whereas arabinose represents the protein signal to be detected.
  • serine integrase Bxb1 and its recombination sites are constructed on the same plasmid to avoid potential trouble of co-transformation.
  • Before induction by arabinose, EGFP is transcribed by promoter OR2+OR1. In contrast, after the induction, serine integrase is expressed to invert the promoter sequence flanked by attB and attP sites, thus expressing mCherry.
Characterization

In order to examine the inversin efficiency of serine integrase, we applied two assays: quantitative RT-PCR and fluorescence spectrophotometry.

  • Quantitative RT-PCR:
    We intended to apply qPCR to measure the ratio of inverted sequence. The primers used in qPCR were designed as shown in Fig. 20. Only after inversion would the primers be in opposite directions to amplify the sequence in between.

Skematic diagram of primers used in qPCR to test the inversion efficiency of serine integrase
Fig 20. Skematic diagram of primers used in qPCR to test the inversion efficiency of serine integrase.

  • Fluorescence spectrophotometry:
    We intended to apply fluorescence spectrophotometry to detect the fluorescence intensity of the two reporter proteins by microplate reader. The expected result is that the fluorescence intensity of mCherry will increase significanly after induction.

pLogic2 for cro/cI characterization

Construction

Plasmid design of pLogic2 for cro/cI characterization.
Fig 21. Plasmid design of pLogic2 for cro/cI characterization.

  • pBAD/araC mimics the TCS promoter PmrB, whereas arabinose represents the protein signal to be detected.
  • Before induction by arabinose, cI is constitutively expressed, inhibiting the expression of reporter gene EGFP. After induction, cro will be expressed to derepress the inhibition caused by cI, thus allowing the expression of reporter gene EGFP.
Characterization

In order to examine the function of cro/cI system, we applied fluorescence spectrophotometry to measure the fluorescence intensity of the reporter protein EGFP by microplate reader. The expected result is that the fluorescence intensity of EGFP will be repressed at low level before induction, but increase significanly after induction.

Construction of Our Integrated Design

Design

After testing the function of each part, we decided to put them together on two plasmids, so that they can be easily co-transformed into bacteria. The designs are shown in Fig 21.


pTCS. pLogicALL.
Fig 21. Schematic diagram of pTCS & pLogicALL.

pTCS has the backbone of pBad, carrying:

  • gene sequence to express nisK and nisR
  • gene sequence to express pmrA and AffiPmrB
  • promoter J23100 driving the expression of nisK, nisR, pmrA and AffiPmrB
  • resistance marker CmR for selection

pLogicALL has the backbone of pBad, carrying:

  • gene sequence to express PnisA, ser integrase, λ repressor, PmrC, cro
  • gene sequence to express mCherry and EGFP
  • promoter Pmr, driving the expression of λ repressor
  • promoter Pr, driving the expression of mCherry and EGFP
  • resistance marker AmpR for selection

    • References

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    [3] Małaczewska, J., & Kaczorek-Łukowska, E. (2021). Nisin-A lantibiotic with immunomodulatory properties: A review. Peptides, 137, 170479.
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    [10] Kotula JW, Kerns SJ, Shaket LA, Siraj L, Collins JJ, Way JC, Silver PA. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc Natl Acad Sci USA. 2014 Apr 1;111(13):4838-43.
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