Part:BBa_K3932000:Design

Arac/pBAD-Proteinase K-glnAp2-TetR-pTet-E7

Assembly Compatibility:

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

  Illegal NheI site found at 1300
  Illegal NheI site found at 2104
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1451
  Illegal BamHI site found at 1239
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1074
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1056
  

Design Notes

We construct a system as follow

1. The synthesis of Proteinase-K starts if our ECN senses arabinose by its pBAD promoter
Processes
• AraC is produced constitutively in control of constritutive promoter which then repressed the pBAD promoter
• In the presence of arabinose, AraC is inactivated and no pBAD repression and Proteinase-K is produced
2. The secretion of Proteinase-K via E7 lysis protein starts if our ECN sense high ammonium level
Processes
• In low ammonia level, activated NtrC induce the glnAp2 promoter to start TetR synthesis
• TetR represses the pTet which results in repression of E7 synthesis
• In high ammonia level, NtrC is inactivated, so no TetR is produced to repress pTet
• Unrepressed pTet promotes transcription of E7 and lysis is occured

Figure 1. Biofilm dispersion system
The system detail are as follow:
Induction
The synthesis of proteinase-K is done in an inducible manner. Constitutive synthesis is counterproductive as we are projecting our product to be delivered in the form of dairy products and a continuous production will burden the system and pose significant problems in packaging and storage. Some things to be considered in the induction system are listed below:

1. the Proteinase-K is needed to be produced and worked before the AMP secretion
2. the Proteinase-K production is inducible and not autoinduced
3. the Proteinase-K has to achieve a targeted optimal concentration for biofilm dispersion before being administered

Acknowledge the aforementioned list regarding the induction system, we choose to use arabinose-induced pBAD promoter production. This is not based merely on the commonly used inducible promoter. We are projecting our product to be available in dairy product drinks, so it is important that our inducer is safe and well tolerated. Arabinose has no observed negative health effects and is even known to improve glycemic control.^18,19 Further packaging design will be discussed in the Implementation section.

Secretion
After the synthesis, the next step is secretion. Due to the periplasmic subcellular location of Proteinase-K²⁰, we design the secretion system via autolysis of our E. coli so we are sure that the Proteinase-K is secreted completely. The E7 lysis protein is chosen not only because of its popularity among iGEM-ers, but the latest study shows the effectiveness of E7 lysis protein in ECN for secreting protease.21 Since we have designed that the Proteinase-K optimal concentration has been achieved, it can be released immediately as it reach the gaster which is occupied by H. pylori. The urease activity of H. pylori converts urea to ammonia. So we find a significantly higher amount of ammonia and sense it via ammonia sensing promoter glnAp2.

Experiment protocol

Detection and characterization of Prot-K

• Aim: to assess how much Proteinase-K (concentration) produced in n-time after induction by arabinose
• Experiment process
  Before testing the function of our ECN synthesized Proteinase-K against the H. pylori biofilm, identification of its presence is important. LB medium is used for expression. All experiments are performed in triplicate.
  
  1. Induction
     The synthesis of Prot-K is induced by 0.1 mM, 1 mM, and 10 mM of arabinose.
  2. Incubation
     Incubation time is set for (1) short period: 10, 20, and 30 minutes and (2) long period: 4, 8, 12, and 24 hours at 4 and 25^oC.¹
  3. Purification
     For purification purposes, 6x His-tag has been attached on the 3’-end of Proteinase-K coding sequences, then Ni-NTA purification is done.
  4. Detection and concentration count
     confirmation of the presence of Prot-K is done by SDS-PAGE. For concentration determination, concentration of purified Proteinase-K is quantified using a NanoDrop.
• Analysis of outcomes
  The concentration of Proteinase-K 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 from each temperature are selected for further analysis. Statistical analysis will be performed using SPSS 24.0.
• Expected response after experiment
  
  1. concentration of produced Proteinase-K in specific incubation time after induction can be used as a parameter to adjust the number of bacteria to be administered so it achieves the optimal amount of Proteinase-K for dispersing biofilm which is known to be 25 μg/ml from literature.2
  2. data from numerous concentration of arabinose and its incubation time in correlation to the concentration of Prot-K produced is valuable to choose whether the product is more convenient to be consumed in a relatively short period of time after mixture (adding arabinose) i.e 10-30 minutes, or the patients need to mix it for the upcoming dose i.e 4-24 hours.
• Parts and Plasmid construction
  
  • BBa_K3932006: araC/pBAD-Proteinase K
  • Plasmid: pM2s2TsR
  • NcoI restriction site (in mRFP1, located at 6,322 bp downstream)
    
  • Primer used for Gibson Assembly:
    

    Forward	ccggttatgcagaaaaaaacTCACACTGGCTCACCTTC
    Reverse	tcggtggaagcttcccaaccAAATAATAAAAAAGCCGGATTAATAATCTG

H. pylori biofilm preparation

• The H. pylori biofilm formation is prepared as the aforementioned protocol in liquid (quantitative) and agar medium (qualitative). All experiments are performed in triplicate. Treatment groups are prepared as follow:
• Negative control: culture medium with H. pylori without Proteinase-K treatment
• Positive control: culture medium with H. pylori added with Proteinase-K (Sigma Aldrich)
• Treatment group: culture medium with H. pylori added with purified Proteinase-K treatment
• Blank: culture medium without H. pylori
• Identification of biofilm
  Biofilm identification is done using crystal violet staining. Observation of optical density is done on a wavelength of 590 nm (OD590) over 12, 24, and 48 hours, in 37^oC using liquid medium. For agar medium, qualitative assessment is done. All experiments are performed in triplicate.

Proteinase-K dispersing function test

• Aim: to assess how much the biofilm is dispersed after exposure to determined Proteinase-K concentration in specific time
• Experiment process
  All experiments are performed in triplicate.
  
  1. Proteinase-K addition
     H. pylori biofilm stained by crystal violet is added by Proteinase-K in the form of
  • purified Proteinase-K from ECN expression (three highest concentration groups in each temperature)
  • Proteinase-K 25 μg/ml (Sigma) as positive control
  2. Activity assessment
     Observation is done in OD590 over 2, 4, 8, 24 and 48 hours in 37oC to assess the dispersion rate of biofilm.
  3. Activity after low pH exposure assessment
     The treatment group that gives the most significant OD reduction in the first 4 hours (because based on the modelling, our AMPs are secreted after 4 hours) is chosen for further activity assessment in different pH levels. The Proteinase-K undergoes 30 minutes of low pH exposure (1, 2, 3, and 4) using adjusted 2 mL HCl (this represents exposure of Prot-K in stomach lumen prior to actively working in the mucosa). Proteinase-K is separated from the HCl solution and retested for its activity.
• Analysis of outcomes
  The outcome of OD will be plotted, compared between observation periods and incubation time of Prot-K expression, unpaired. ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis will be done. Statistical analysis will be performed using SPSS 24.0.
• Expected response after experiment
  
  1. Residual activity is known in the environment exposed to low extreme pH and temperature of 37oC which are the physiological condition of human stomach
  2. Residual activity of Prot-K will affect the degradation of antimicrobial peptide (AMP) PGLa-AM1, which affect the bactericidal activity of AMP
  
  

E7 lysis function

• Aim: to assess the time needed to lysis ECN via E7 expression after ammonium induction.
• Experiment process
  As the E7 expression in ECN has been proved to effectively result in autolysis, here we assess how much time is needed to lysis the ECN via E7 expression that is 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 E7 lysis transformed ECN and without lysis protocol.
  • Positive control: LB culture medium without E7 lysis transformed ECN and with lysis protocol.
  • Treatment group: LB culture medium with E7 lysis 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: culture medium without ECN
  1. Induction
     The synthesis of E7 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 30 minutes, 1, 2, 3, and 4 hours at 37^oC
     
  3. Lysis Assessment
     Serial documentation of OD600 is done after respective incubation time. Standard curve is made using iGEM calibration protocol Conversion of OD600 to CFU/ml. OD600 then is converted to CFU/mL.
• Analysis of Outcomes
  The outcome of CFU will be plotted, compared between incubation periods and induction concentration, unpaired. ANOVA and Post Hoc (normal distribution) or Kruskal-Wallis and Mann-Whitney (non-normal distribution) analysis will be done. Statistical analysis will be performed using SPSS 24.0.
  
• Expected response after experiment:
  • optimal concentration of ammonium that triggers the highest lysis activity is an important information to determine how long the system will work and how fast is the rate of Prot-K secretion. Since arabinose may be “washed” from the stomach while Prot-K is relatively more stable and resilient in the human stomach, considering to synthesis enough amount of Prot-K since the beginning by adjusting the number of bacteria, amount of arabinose for induction, and in a relatively short time of incubation (consider the consumer convenient) before administration is important.
  • The concentration of ammonium surrounding the H. pylori is unknown. A study examined the urease activity from H. pylori. In an acidic environment, optimally at pH of 3, as much as 10⁹ CFU/mL H. pylori are viable, the urease activity is documented as 204 μmol NH3/min/mg protein. If converted to w/v it is approximately 0.3% of ammonium.⁴
  • Considering the average amount of H.pylori in active infection reaches up to 10⁶ CFU (in a certain biopsy location)⁵, the amount of ammonium produced by the urease is lower than 0.005% (baseline experiment concentration). But, several data shows that a lower concentration (0.02 fold of 0.005%) just needs an extra 30-60 minutes for product amount to rise significantly.
  • However, if even the highest concentration of ammonium (0.5% w/v) does not exhibit a satisfied response and produce enough E7, neither enable the release of Prot-K in desirable rate, addition of lysis protein or transcription enhancer may be needed. From another point of view, the low secretion rate of Prot-K may be beneficial as a model of extended release drugs.
Parts and Plasmids Construction
• BBa_K3932007: glnAp2-TetR-pTet-E7
• Plasmid pM2s2TsR
• NcoI restriction site (in mRFP1, located at 6,322 bp downstream)
  
• Primer used for Gibson Assembly:
  

  Forward	ccggttatgcagaaaaaaacTAGATGCCTCCACACCGC
  Reverse	tcggtggaagcttcccaaccAAATAATAAAAAAGCCGGATTAATAATCTGGC