Part:BBa_K3932011:Design

pCopA-His6-SUMO-PGLaAM1-TetR-pTet-cI-pcI- Ulp1-Lysis

Assembly Compatibility:

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

  Illegal NheI site found at 2490
  Illegal NheI site found at 4585
  Illegal NheI site found at 4608
  Illegal NotI site found at 3186
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 372
  Illegal BamHI site found at 3155
  Illegal XhoI site found at 3164
  Illegal XhoI site found at 3195
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 557
  Illegal AgeI site found at 4190
  Illegal AgeI site found at 4260
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 4841
  

Design Notes

We construct a system as follow

1. The synthesis of SUMO-PGLa-AMI starts if our ECN senses the presence of ammonia
Processes
• Ammonium ions produced by H. pylori urease
• NH4+ induces pCopA, expressing SUMO-PGLa-AM1 and TetR genes downstream
2. The activation of PGLa-AM1 via cleavage of SUMO-PGLa-AM1 by the Ulp-1 protease that is controlled by a TetR-based timer cassette
Processes
• TetR represses pTet further downstream
• cI expression, which was under pTet, is inhibited
• After a time constant (see Modelling), extant cI to degrades
• In turn, cI repression of pcI is inhibited
• Ulp-1 is expressed and cleaves the SUMO from present SUMO-PGLa-AM1
• PGLa-AM1 attains a biologically active structure without SUMOylated terminal
3. The secretion of activated PGLa-AM1 via holin-antiholin lysis system
Processes
• pcI activates via cI (repressor) degradation
• Holin and endolysin are expressed
• Holin forms pores in the cell membrane
• Extant antiholin, constitutively expressed, halts holin action to provide another time delay for PGLa-AM1 accumulation
• Endolysin and other intracellular enzymes degrade the cell wall
• Host is lysed and cell contents released into environment
• Cell contents including activated PGLa-AM1 which also further enhances the kill switch function

Figure 1. H. pylori eradication system
The system details are as follow
Induction
First of all, synthesis of an active AMP inside a bacteria will negatively affect the host (our E. coli) as PGLa-AM1 is also toxic to E. coli. Although the MIC for E. coli is extremely higher than for H. pylori (51.76) So there is a huge chance of our synthesized AMP causing the host to self-terminate. Unfortunately, if that happens we are unable to control the production rate or concentration and at the end we are unable to kill the H. pylori (instead just killing our E. coli). So, we are designing the AMP to be inactivated by a well known fusion system using SUMO peptide which incidentally also increases its solubility. The Peptidor project from iGEM TU-Delft 2013 inspired us a lot in developing this system.⁷

There are two main things to be considered for designing our induction system

1. This system must work after the biofilm dispersion happens
2. The production of FAMP is induced by the presence of H. pylori
3. The production of FAMP is inducible and not autoinduced

To remind you, our Proteinase-K is being secreted as the E. coli detect ammonia - H. pylori is producing ammonia as a product of urea metabolism by urease -. It is not possible to use AI-2 (although it is widely used by several iGEM teams) as an inducer due to the fact that the E. coli is naturally secreting the same molecule (except if LuxS mutation is done).8 This will lead to a premature synthesis of FAMP because of the auto inducible production. Only ammonia can be used as a signal in this setting. We choose the ammonia inducible promoter (previously known as copper sensing promoter), pCopA.⁹

Activation
As we use ammonia sensing in the Proteinase-K secretion and FAMP production system, these will happen at the same time. To buy enough time for the Proteinase-K to disperse a significant amount of biofilm and to produce enough FAMP (targeted concentration), the timer cassette is set. To activate our FAMP: SUMO-PGLa-AM1, we are adapting the Peptidor system. The system involves double repressible promoters (pTet and pcI), aiming to have enough time for FAMP production. The last component of the activation system is to cleave the FAMP using Ulp-1 protease which acts specifically as SUMO protease.

Secretion
The last step is absolutely secreting the activated AMP. Still, we are benchmarking the Peptidor project as their mathematical model is quite well developed for adaptation. The holin-antiholin system is used for our kill switch mechanism, controlled under the pcI. The lysis of our bacteria is not only for secretion purposes, but also to make sure that our engineered bacteria, although served as a probiotic strain that is widely accepted as safe species, does not pose further risk of mutation and become pathogenic.

Experiment protocol

Detection and characterization of SUMO-AMP

• Aim: to assess how much SUMO-AMP produced in n-time after induction by ammonium in different concentrations.
• Experiment Process
  
  1. Induction
     The synthesis of SUMO-AMP is induced by ammonium hydroxide 0.005% (w/v), 0.01 fold, 0.1 fold, 10 fold, and 100 fold concentration
  2. Induction
     Incubation time is set for 2, 4, 6, and 8 hours at 37oC using a shaker incubator.
  3. Purification
     A 6x his-tag is attached to the 5’-end of SUMO-AMP structure, thus Ni-NTA purification is done.
  4. Detection and concentration count
     The solution is put at the SDS-PAGE for further characterization. Concentration of purified SUMO-AMP is measured using a NanoDrop.
• Analysis of outcomes
  The concentration of SUMO-AMP in different environments are compared statistically using ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis. Three highest concentration groups are selected for further analysis. Statistical analysis will be performed using SPSS 24.0.
• Expected response after experiment
  
  1. concentration of produced SUMO-AMP in a specific timestamp after induction in response to several different concentrations of ammonium can be used as a parameter to adjust the number of bacteria to be administered so it achieves the optimal amount of SUMO-AMP as the precursor of active AMP (PGLa-AM1).
     • PGLa-AM1 MIC from in vitro study is 1μg/ml
     • PGLa-AM1 optimal dose from in vivo (murine) study is 60 mg/kg
  2. The optimal response corresponding to the inducer (ammonium) concentration is valuable information to further reevaluate the inducer system. This also correlates with how much ammonia that is available to be sensed from H. pylori production. If higher concentrations are needed, an enhancer of transcription may be necessary. We discuss more detail regarding the ammonium concentrations in the real world (stomach mileu) below.
  
  Parts and Plasmid Construction
  • BBa_K3932008: pCopA-His6-SUMO-PGLaAM1
  • Plasmid pM2s2TsR
  • NcoI restriction site (in mRFP1, located at 6,322 bp downstream)
    
  • Primer used for Gibson Assembly:

  Forward	ccggttatgcagaaaaaaacCCTTTTTATAGATGCGGG
  Reverse	tcggtggaagcttcccaaccAAATAATAAAAAAGCCGGATTAATAATC

Detection and characterization of Ulp-1

• Aim: (1) To determine how much Ulp1 is expressed in a given time after induction by ammonia, (2) to determine the time delay of timer cassette.
• Experiment process
  
  1. Induction
     The synthesis of Ulp-1 is induced by ammonium hydroxide 0.005% (w/v), 0.01 fold, 0.1 fold, 10 fold, and 100 fold concentration.
  2. Incubation
     Incubation time is set for 1, 2, 3, and 4 hours at 37 ^oC using a shaker incubator.
  3. Purification
     For purification purposes 6x his-tag is attached to Ulp-1 protein, thus Ni-NTA purification is done.
  4. Detection and concentration count
     The solution is put at the Tricine SDS-PAGE for further characterization. Concentration of purified SUMO-AMP is measured using a NanoDrop.
• Analysis of outcomes
  The concentration of Ulp-1 in different environments are compared statistically using ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis. Three highest concentration groups are selected for further analysis. Statistical analysis will be performed using SPSS 24.0.
• Expected response after treatment:
  
  • The difference in incubation time would provide insights in Ulp-1 expressions over time. The different amount of expressed Ulp-1 is used to determine how many SUMO-AMP each can cleave.
    
    • 1/1000 ratio of Ulp1/SUMO can cleave SUMO tag 95% after 12 hours.
  • The amount of Ulp1 received is valuable as a parameter to measure the time needed to express sufficient Ulp-1 concentration. The time can be adjusted to express Ulp necessary for a sum of SUMO-AMP expressed at a specific time. The amount of expressed Ulp1 can be adjusted by its incubation time if necessary. This also will lead to adjustment of the timer cassette that can be shortened to produce enough Ulp-1 for SUMO-AMP cleavage.

Parts and Plasmid construction

• BBa_K3932009: pCopA-TetR-pTet-cI-pcI-Ulp1
• Plasmid pM2s2TsR
• NcoI restriction site (in mRFP1, located at 6,322 bp downstream)
  
• Primer used for Gibson Assembly:

Forward	ccggttatgcagaaaaaaacCCTTTTTATAGATGCGGG
Reverse	tcggtggaagcttcccaaccAAATAATAAAAAAGCCGGATTAATAATC

Ulp-1 protease & PGLa-AM1 detection and characterization

• Aim: to assess how much SUMO-AMPs are cleaved and PGLa-AM1s are activated in a correlated time.
• We mix the expressed Ulp-1 and the expressed SUMO-AMP complex in a buffer (50 mM Tris/HCl, pH 8.0, 0.1M NaCl, 10mM DTT) at 25 ^oC, and 37 ^oC for 4, 6, 8, 12, and 24 hours. We calculate PGLa-AM1 concentration with NanoDrop. PGLa-AM1 is characterized and analyzed using Tricine SDS-PAGE with PageBlue staining.
• Analysis of outcomes
  The concentration of PGLa-AM1 from paired SUMO-AMP and Ulp-1 (e.g both from 1 hour induction) are compared statistically using ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis. Three highest concentration groups are selected for further H. pylori eradication tests. Statistical analysis will be performed using SPSS 24.0.
• Expected results after treatment:
  
  • The amount of successfully cleaved PGLa-AM1 is needed to measure the necessary expression of SUMO-AMP and corresponding Ulp1 over different time and temperature.
  • This test is also necessary to determine whether or not the amount of PGLa-AM1 produced is enough to kill a given amount of H. pylori at a given time of Ulp1 and SUMO-AMP expression. The amount of Ulp1 expression may be increased to cleave all SUMO-AMP complexes faster.

Kill switch function test

• Aim: To measure the time it took for induced E. coli cells to lyse over time after the induction by ammonium.
• Experiment process
  
  Here we assess how much time is needed to lysis the ECN via the holin-antiholin-endolysin (kill switch system) expression under the control of ammonia sensing promoter glnAp2. All experiments are performed in triplicate. Treatment groups are prepared as follow
  
  • Negative control: LB culture medium without kill switch parts being transformed ECN and without lysis protocol.
  • Positive control: LB culture medium without kill switch parts being transformed ECN and with lysis protocol.
  • Treatment group: LB culture medium with kill switch parts being transformed ECN and without lysis protocol induced by ammonium hydroxide 0.005% (w/v)³ 0.01 fold, 0.1 fold, 10 fold, and 100 fold.
  • Blank: LB culture medium without ECN.
  1. Induction
     The kill switch system is activated by the induction using ammonium hydroxide 0.005% (w/v), 0.01 fold, 0.1 fold, 10 fold, and 100 fold concentration.
  2. Incubation
     Incubation time is set for 2, 4, 6, and 8 hours at 37 ^oC using a shaker incubator.
  3. Activity assessment
     Serial documentation of OD600 is done after respective incubation time. Conversion of OD600 to CFU/ml is done using iGEM calibration protocol.
• Analysis of outcomes
  The OD in different environments are compared statistically using ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis. Statistical analysis will be performed using SPSS 24.0.
• Expected results after treatment:
  
  • This test is necessary to know the time it takes for the E. coli to lyse itself. With this test, one can know if the necessary proteins (PGLa-AM1) are expressed enough before the lysis takes place. If the optimal amount of PGLa-AM1 is not achieved, synthesis intensification is needed using an enhancer or more bacteria is needed.
  • The kill switch promotor can be induced less or more, to adjust the AMP secretion rate.

Parts and Plasmid construction

• BBa_K3932010: pCopA-TetR-pTet-cI-pcI-kill switch
• Plasmid pM2s2TsR
• NcoI restriction site (in mRFP1, located at 6,322 bp downstream)
  
• Primer used for Gibson Assembly:

Forward	ccggttatgcagaaaaaaacCCTTTTTATAGATGCGGGAGG
Reverse	tcggtggaagcttcccaaccGAGAGCGTTCACCGACAAAC

H. pylori culture preparation

• Growth is preferable to a system atmosphere of 10% CO2, 5% O2, and 85% N2 (Oxoid Campygen CN0025). We use H. pylori of strain ATCC 49503. We use two types of medium depending on the analysis:
  • Solid medium is prepared using columbian blood-based agar.
  • Liquid medium is prepared using brucella broth.
  • For non biofilm forming culture, 2 mg/mL N-acetylcysteine is added.
• Expected results after treatment:
  
  • This protocol should give the idea of the best medium to culture with different analysis. Liquid medium, for example, is better used when we want to count cells via absorbance.
  • The results of this experiment should also provide us data on the viability of the chosen mediums to compare if a necessary junction appears to change mediums for a more optimised system.
  • This protocol also gives us the exact number of starting viable cells before we count them again after addition of PGLa-AM1.

PGLa-AM1 function test

• Aims: to determine the bactericidal activity of produced PGLa-AM1 (concentration from the purification) to H. pylori
• Experimental process
  Brucella broth with different concentrations of PGLa-AM1 (according to incubation time) is prepared in a microaerophilic environment at 37 ^oC. H. pylori cells are added to the various PGLa-AM1 concentrations and dilute them periodically at 0, 15, 30, 45, 60 minutes. The diluted cultures were incubated before measuring the number of viable CFU/mL.
• Analysis of outcomes
  The OD in different PGLa-AM1 concentration treatment and dilution time are compared statistically using ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis. Statistical analysis will be performed using SPSS 24.0.
• Expected response after treatment:
  
  • The number of viable H. pylori available over incubation time after mixing with PGLa-AM1 can give insights on the activity of different amounts of PGLa-AM1 to kill H. pylori. This parameter can be used to determine the necessary amount of PGLa-AM1 and the time needed to fully eradicate H. pylori. This is also important information about how many doses are needed to fully eradicate H. pylori since H. pylori keep reproducing while they got killed.
  • The number also correlates with the amount of E. coli cells, and its induction time needed to produce adequate amounts of PGLa-AM1. This can be achieved by increasing the number of E. coli cells, increasing the induction activity for protein expression, or lowering the kill-switch timer.
  • The resultant MIC gives us a baseline to adjust the PGLa-AM1 production by its expression and activation (in tandem with results from experiments described previously) as a starting point to tweak the final output of the system.
  • The bactericidal graph allows us to tune the final genes using results from previous experiments in order to produce the optimum amount of PGLa-AM1 by elucidating the relative effect over time the PGLa-AM1 production rate has on the target pathogen.
  • Similarly, it also allows us to optimize the system output to prevent toxicity to our host organisms (humans and delivery bacteria both) while still retaining satisfactory results.
  • Estimation of the relative effect per time will be correlated with production rate over time to produce a dose effect graph for humans for PGLa-AM1 which, while not directly useful to our project, is novel information for future projects within or without iGEM.

Proteinase-K and PGLa-AM1

• Aims: to determine the residual activity of produced PGLa-AM1 after exposure to Proteinase-K
• Experimental process
  As Proteinase-K will cleavage our AMP, it is necessary to do an experiment of this cleavage and inactivation of PGLa-AM1by Proteinase-K. The optimal PGLa-AM1 concentration from the previous experiment and its parallel Proteinase-K production (from the same condition: ammonium concentration, temperature, and incubation time) are mixed and proceed to the PGLa-AM1 function test.
• Expected response after treatment
  
  • The degree of which proteinase K affects PGLa-AM1 function given a certain time. This insight will allow us to assess the needed rates of production required to overcome proteinase K hydrolysis of our functional protein. This upper limit of proteinase K production and lower limit of PGLa-AM1 production will allow us maximise biofilm degradation whilst maintaining proper antibacterial function.
  • Conversely, we also need to determine the lower limit of proteinase K concentration to still provide effective biofilm degradation without significantly impairing PGLa-AM1 action. This would also allow us to determine the upper limit of PGLa-AM1 production before it can overcome proteinase K inhibition. This would allow us to precisely engineer a rate that produces exactly as much as needed as opposed to grossly overshooting the PGLa-AM1 production rate in an effort to blindly counter proteinase K action.