iGEM 2019 — Team Technion-Israel

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# Team:Technion-Israel/Notebook

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- [Safety](https://2019.igem.org/Team%3ATechnion-Israel/Safety)
- [Judging](https://2019.igem.org/Team%3ATechnion-Israel/Judging)

Week 1 (May 5-11)

* + Choosing the topic of our project
  + Gathering essential information for our project
  + Planning future timeline
  + Planning the experiments needed for the proof of concept
  + Reviewing protocols

Week 2 (May 12-18)

* + Choosing the name and the logo of the project
  + Researching the standard properties and content of honey
  + Contacting beekeepers and honey experts
* + Research of the essential enzymes and their sources, as well as their assays
* + Initiating the planning of a Hackathon-like event
* + Organizing the equipment in the lab

Week 3 (May 19-25)

* + Dividing the team into sub teams
  + Sending requests for sponsorships
* + Looking for the gene sequences of the wanted enzymes as well as their sources, assays and mechanism

Week 4 (May 26- June 1th)

* + Planning experiments on industrial enzymes
  + Writing protocols for reducing sugar assays, determination of acidity, moisture and sucrose
  + Finding the most similar commercial enzymes for in-vitro experiments
  + Initial test for sucrose degradation using different combinations of glucose oxidase, invertase, and catalase. As the pH meter was malfunctioning, the process needs to be repeated. Moreover, bigger time intervals should be taken, and the test needs to be continued for more than 5 hours at the first phase.
* + Adjusting the optimized gene sequence for *B.Subtilis* to match DTI's requirements
  + Designing a few optional circuits for hydrogen peroxide-based inhibition of Glucose Oxidase and analyzing them

Week 5 (June 2-8)

* + Safety form draft
  + Project description and project inspiration draft
  + Researching possible applications for our product
* + Participating in the Vegan TLV Festival
* + Honey standard assays were performed on commercial honey - determination of moisture and acidity. The results will be used as a reference to our final honey product
  + ![]( https://static.igem.org/mediawiki/2019/1/17/T--Technion-Israel--Moisture_6_6.png)

    ***Figure 1:*** Refractometer and commercial honey tested, 6.6.19

    + ![](https://static.igem.org/mediawiki/2019/a/a7/T--Technion-Israel--Acidity_6_6.png)

      ***Figure 2:*** Acidity determination by titrartion on dilute commercial honey tested, 6.6.19
    + pBE-S transformation to  *E. coli*  Top 10
    + Glycerol stock for  *E. coli*  Top 10 + pBE-S
    + Mini prep for pBE-S
    + Nano drop results:

      |  | pBE-S 1 | pBE-S 2 | pBE-S 3 | pBE-S 4 | pBE-S 5 |
      | --- | --- | --- | --- | --- | --- |
      | Concentration [ng/μl] | 825.7 | 706.1 | 733.0 | 703.0 | 689.3 |
      | 260/280 | 1.80 | 1.83 | 1.82 | 1.83 | 1.81 |
      | 260/230 | 2.22 | 2.25 | 2.23 | 2.26 | 2.22 |
    + Digestion of pBE-S using Mlu1-HF and Eag1-HF
    + Gel electrophoresis for the cut pBE-S
    + Clean-up of the reaction products
    + Nano drop results:

      |  | 1 | 2 |
      | --- | --- | --- |
      | Concentration [ng/μl] | 24.9 | 30.4 |
      | 260/280 | 2.61 | 2.55 |
      | 260/230 | 0.73 | 0.74 |
      | Final Elution Volume [μl] | 30 | 35 |
    + Experiments on commercial honey: pH, humidity, acidity
* + Designing the general synthetic circuit
  + Researching parts which react to hydrogen peroxide
  + Colony PCR primer design, specific for signal peptides

Week 6 (June 9-15)

* + BGU meetup
* + Reducing sugars practice experiment
  + Preparing protocols for cloning of gBlocks
* + Developing a general model scheme
  + Meeting with Roee Amit
* + Researching the option of using Cello
  + Researching pH dependent circuits

Week 7 (June 16-22)

* + Meeting with Alexey Tomsov former iGEM participant and judge, currently director in the sustainable food industry, who gave us valuable insights and advice to better define our project
* + Looking for the coefficients of each enzyme
  + Looking for the appropriate equations to describe the enzyme activity
* + Repeating the digestion of pBE-S using Mlu1-HF and Eag1-HF restriction enzymes
  + Colony PCR for  *E. coli*  Top 10 with pBE-S and gel electrophoresis for the colony PCR products
  + Clean-up of pBE-S after RE reaction from gel. Final concentration: 13.5 ng/µl
  + Digestion of signal peptide genes: AmyE, SacB, SacC
    Final concentration: 17.1, 12.8 and 11.5 ng/µl accordingly
* + Settling on the parts needed for our project: a PerR-based promotor, an inhibitor gene – such as LacI and the promotor which it inhibits (pLac), and finally – Glucose Oxidase

Week 8 (June 23-29)

* + Suspension of gBlocks of the signal peptide genes, as well as the genes of invertase and glucose oxidase
  + Restriction of the signal peptide genes using Mlu1-HF and Eag1-HF and purification of restriction products using Promega Kit. Elution has been performed with 20 microliters of Ultra-Pure Water
  + Nano drop results:

    |  | AmyE | SacB | SacC |
    | --- | --- | --- | --- |
    | Concentration [ng/μl] | 16.3 | 12.4 | 10.0 |
    | 260/280 | 3.36 | 2.32 | 2.42 |
    | 260/230 | 0.53 | 0.51 | 0.52 |

Week 9 (June 31- July 6)

* + Ligation to pBE-S with different signal peptides: AmyE, SacB, SacC
  + Transformation to  *E. coli*  Top 10
  + ![](https://static.igem.org/mediawiki/2019/7/7b/T--Technion-Israel--plates_4_7_19.png)

    ***Figure 3:*** *E. coli* Top 10 + pBE-S + Signal Peptides(SP), rest, 4.7.19

Week 10 (July 7-13)

* + Colony PCR for the  *E. coli*  Top 10 colonies using insert specific primers for the three signal peptides
  + ![](https://static.igem.org/mediawiki/2019/5/56/T--Technion-Israel--gel_7_7.png)

    ***Figure 4:*** Gel electrophoresis of pBE-S + SP, 7.7.19
  + Mini prep of pBE-S + Signal peptides and sequencing

Week 11 (July 14-20)

* + Reverse PCR of pBE-S + Signal peptides
  + Gel electrophoresis of PCR products
  + ![](https://static.igem.org/mediawiki/2019/9/90/T--Technion-Israel--gel_18_7.png)

    ***Figure 5:*** Gel electrophoresis of pBE-S + SP, 18.7.19
  + Clean-up of PCR products
  + Nano drop results:

    |  | pBE-S + AmyE | pBE-S + SacB | pBE-S + SacC |
    | --- | --- | --- | --- |
    | Concentration [ng/μl] | 95.4 | 83.3 | 100.7 |
    | 260/280 | 1.78 | 1.74 | 1.79 |
    | 260/230 | 0.87 | 1.41 | 1.09 |

Week 12 (July 20-27)

* + Initial cloning of the Glucose Oxidase and Invertase gBlocks into the pBE-S plasmids with different signal peptides. The cloning was done using the Gibson Assembly method; however, the process was done using close plasmids instead of plasmids which were opened via Reverse PCR. Therefore, the process needs to be repeated
  + As there were not enough materials needed for cloning, the process was repeated with few adjustments. The insert to plasmid ratio was 1:1 instead of 3:1
  + Transformation into  *E. coli*  Top 10
  + ![](https://static.igem.org/mediawiki/2019/8/81/T--Technion-Israel--plates_23_7.png)

    ***Figure 6:*** *E.coli* top 10 + pBE-S + GOx/Invertase, 23.7.19

Week 13 (July 28-August 3)

* + Colony PCR for  *E. coli*  Top 10 colonies using primers specific to the inserts. At first, the PCR program was not set on hold mod at 4⁰C at the end, therefore the samples might have been damaged. The process was repeated, and the results were still negative
  + ![](https://static.igem.org/mediawiki/2019/3/34/T--Technion-Israel--gel_31_7.png)

    ***Figure 7:***  Colony PCR for pBE-S + GOx/Invertase, 31.7.19
  + Reverse PCR for the pBE-S + signal peptides in order to have a sufficient amount of plasmid, gel electrophoresis and clean-up from gel
  + Amplification of the gBlocks of the enzyme genes using PCR and gel electrophoresis. We have used an insufficient amount of Q5 enzyme accidently, and therefore the PCR was not successful and needs to be repeated
  + Transformation of pBE-S + Signal peptides into  *E. coli*  Top 10
  + Tests for sucrose degradation using different combinations of glucose oxidase, invertase, and catalase. The pH measurements were better but shorter time intervals should be taken at the beginning of the process. Moreover, the sugar content needs to be examined
* + Specific sequences were chosen for the synthetic circuit parts: katA, pLac and LacI
  + The effect of hydrogen peroxide on katA (PerR regulon promoter) was researched
* + Perfecting the scheme of modeling the entire process done by the main enzymes
  + Looking for coefficients for secretion as well as the kinetic coefficients for the enzymes
* + Working on the iGEMthon – meeting with Haifa municipality, contacting relevant companies and looking for potential lecturers
  + One Minute iGEM Challenge – filming and editing our one-minute video as well as the invitation video

Week 14 (August 4-10)

* + After debating whether the honey-like product needs to for food or medical applications, the chosen application is food
  + Working on the Wiki design
* + Growth coefficient was found, as well as kinetic coefficients for invertase
  + Developing the correct equations
  + Coding in Matlab
* + The main parts of the synthetic circuit were planned and ordered as gBlocks, along with the appropriate primers
* + Reverse PCR for the pBE-S + signal peptides, followed by gel electrophoresis and clean-up from gel. The volume that was loaded is higher than usual, so the comb was typed with cellotape to create wider wells. The PCR products were cleaned from gel+ Nano drop results:

      |  | pBE-S + AmyE | pBE-S + SacB | pBE-S + SacC |
      | --- | --- | --- | --- |
      | Concentration [ng/μl] | 135.4 | 109.9 | 159.9 |
      | 260/280 | 1.9 | 1.92 | 1.9 |
      | 260/230 | 0.36 | 0.16 | 0.33 |

      + Amplification of gBlocks– invertase and glucose oxidase – via PCR. the process was repeated a few more time as the melting temperature was not ideal for the process. The PCR products were cleaned from gel+ Nano drop results:

          |  | GOx | Invertase |
          | --- | --- | --- |
          | Concentration [ng/μl] | 52.0 | 92.2 |
          | 260/280 | 1.94 | 1.93 |
          | 260/230 | 0.29 | 0.27 |

          + The Invertase and Glucose Oxidase genes were cloned to pBE-S + different signal peptides. The products of reaction were transformed into  *E. coli*
          + Tests for sucrose degradation using different combinations of glucose oxidase, invertase, and catalase. Alongside pH tests, the glucose levels were tested throughout the experiment. Honey-like sugar concentrations were achieved after 3 days, and the gluconic acid decreased the pH rapidly
          + Performing activity test to plot a calibration curve for invertase. Unfortunately, the procedure has failed due to low concentration of picric acid
          + Performing an activity test to plot a calibration curve for invertase. Unfortunately, the procedure has failed due to low enzymatic activity
          + Performing an activity test to plot a calibration curve for invertase, using an increased concentration of enzyme. Unfortunately, the procedure has failed again due to low enzymatic activity
          + Preparing a growth medium modified for making our model bacteria competent – and making sure to supply competent bacteria to our cloning subteam
* + Contacting potential sponsors
  + Meeting with ASAT, the Technion Student Association
  + Final lineup was decided
  + Final Budget

Week 15 (August 11-17)

* + Designing group T-shirts
  + Initial wiki graphics
* + Colony PCR for  **E. coli**  Top 10 colonies using primers specific to the inserts
  + ![](https://static.igem.org/mediawiki/2019/d/d8/T--Technion-Israel--gel_11_8.png)

    ***Figure 8:***  Colony PCR for pBE-S + SP + GOx/Invertase, 11.8.19
  + We decided to continue with the successful colonies, from which we’ve made starters to create glycerol stocks and miniprep. The colonies were sent for sequencing
  + For the pBE-S + AmyE + Invertase colonies the process was not successful, and therefore was repeated
  + ![](https://static.igem.org/mediawiki/2019/e/ea/T--Technion-Israel--plates_13_8.png)

    ***Figure 9:***  Colony PCR for pBE-S + AmyE + Invertase, 13.8.19
  + Again, from the successful colonies we’ve made starters to create glycerol stocks and miniprep. The colonies were sent for sequencing
  + Initial preparation of  **Bacillus subtilis** RIK1285 isolation plates
  + Performing experiments in order to determine the appropriate time needed to run the assay on the secreted invertase enzyme and preparing a calibration curve for each time period that would assist us in deducing the activity of the secreted enzyme
  + Performing catalase activity protocols – did not yield any results
  + Performing an activity test to plot a calibration curve for invertase, using an increased concentration of enzyme. Unfortunately, the enzymatic activity was too high, resulting in overflown absorbance and failure of the experiment
  + Performing an activity test to plot a calibration curve for invertase, using an increased concentration of enzyme. Unfortunately, the enzymatic activity was not detected
  + Performing a test with an appropriate enzyme amount. The test was performed in various times to determine the most suitable time for the invertase assay. 10 minutes were chosen as the ultimate time
  + Performing a growth experiment on *Bacillus subtilis* RIK1285 in L.B medium taking O.D measurements every hour – to determine the growth curve so our team could consider it in other experiments
  + Repeating the L.B growth experiment
* + Meeting with Anat Sheer from Haifa municipality for iGEMthon
  + Setting final details: location, date, number of participants and theme
  + Launching the One Minute iGEM Challenge

Week 16 (August 18-24)

* + Deciding on a track: Standard Track, Food and Nutrition
  + Initial design of wiki set-up, defining future goals and working on wiki content and task segmentation
  + Finding out about the use of SBP capsules to separate the bacteria, reaching out to the developers in order to get samples
* + Analyzing sequencing results. As some of the results were unexpected, re-evaluation of the lab wok timeline was needed, as well as organization of stocks, getting rid of samples with the wrong DNA
  + Transformation of pBE-S + signal peptides + enzyme genes into  **Bacillus subtilis**
  + Turbidity test of general growth ability of native *B. subtilis* RIK1285 on different mediums with different sugar concentration. The conclusion: LB is crucial for the growth of the bacteria
  + Stop codon was removed from the plasmids using dpn1+PNK+ligation
  + Performing activity test to plot a calibration curve for glucose oxidase. Unfortunately, the procedure has failed due to low concentration of ABTS
  + Performing experiments in order to create a calibration curve connecting the amount of glucose oxidase units in the solution and the amount of oxidized glucose, as measured by absorbance (416 nm). Due to saturation, the curve was not successful, and the process should be repeated
  + Performing an activity test to plot a calibration curve for glucose oxidase. The procedure was completed successfully
  + Performing catalase activity test. Unfortunately, the procedure has failed due to lack of hydrogen peroxide present in the enzyme solution. (completely decomposed by the catalase)
  + More biochemical tests analyzing the change of pH during time with different enzyme combination, as well as measuring gluconic acid. The pH levels decrease quick meaning there is not enough time for the enzymes to work
  + Performing a growth experiment on the *Bacillus subtilis* RIK1285.
    This time we have checked thr growth ability in changing concentration of hydrogen peroxide, sucrose, and glucose – taking O.D measurements every hour – to determine the growth curve so our team could consider it in other experiments
  + Repeating the growth experiment - With changing concentration of hydrogen peroxide, sucrose, and glucose.
  + Performing a growth experiment on the team's engineered bacteria, this time checking the growth ability in changing concentrations of hydrogen peroxide
* + Meeting with Anat Sheer from Haifa municipality as well as the Volunteer Coordinator
  + Contacting schools’ excellence coordinators
  + Meeting Noar Socher Mada (via phone call)
  + Recruiting sponsors for the iGEMthon
* + Final equations for the enzymes’ kinetics
  + Meeting with Roee Amit

Week 17 (August 25-31)

* + Template programming for the wiki
  + Arrival of SBP capsules
* + Performing experiments in order to create a calibration curve connecting between the amount of catalase enzyme in the solution and the amount of hydrogen peroxide, as measured by yellow hydrogen peroxide-molybdate complex (405 nm)
  + Performing the glucose oxidase activity tests on the bacterial lysates and supernatants on selected colonies (pBE-S + SacB + Glucose Oxidase 1, pBE-S + SacB + Glucose Oxidase 2, pBE-S + AmyE + Glucose Oxidase 1). No enzymatic activity was detected
  + Continuing the removal of the stop codon from the rest of the plasmids using dpn1+PNK+ligation
  + Performing experiments of  **Bacillus subtilis** growth on LB, using only a small starter (4 ml)
  + Performing tests on lysates and supernatants of  **Bacillus subtilis** to identify enzyme activity, running SDS page gels and Bradford calibration for the first time
  + First try of SDS-PAGE. We ran the samples of SacC Invertase, SacB Invertase, AmyE GOx. Only the standard was visible in the gel so we realized that we need to concentrate the supernatant and lysate
  + Last time preforming growth experiment with changing concentration of hydrogen peroxidase, sucrose, and glucose
* + Simplifying the kinetic equations of the enzymes
  + Researching the effect of pH change over time on kinetics
* + Setting mentor shifts for the iGEMthon, contacting more optional mentors
  + Contacting potential lecturers and setting some of the lectures
  + Sending documents for approval of donations

Week 18 (September 1-7)

* + Mini prep of pBE-S + AmyE + Invertase 1
  + Nano drop results:

    |  | Invertase 1-1 | Invertase 1-2 | Invertase 1-3 |
    | --- | --- | --- | --- |
    | Concentration [ng/μl] | 609 | 588 | 476.1 |
    | 260/280 | 1.84 | 1.84 | 1.86 |
    | 260/230 | 2.18 | 2.81 | 2.13 |
    | Final Elution Volume [μl] | 40 | 40 | 40 |
  + Reverse PCR for the pBE-S + signal peptides, followed by gel electrophoresis and clean-up from gel. The PCR products were cleaned from gel
  + Nano drop results:

    |  | pBE-S + AmyE | pBE-S + SacB | pBE-S + SacC |
    | --- | --- | --- | --- |
    | Concentration [ng/μl] | 410.2 | 430.8 | 432.5 |
    | 260/280 | 1.84 | 1.84 | 1.84 |
    | 260/230 | 2.21 | 2.23 | 2.24 |
    | Final Elution Volume [μl] | 35 | 35 | 35 |
  + Reverse PCR for the pBE-S backbone only, followed by gel electrophoresis and clean-up from gel. The PCR products were cleaned from gel
  + ![]( https://static.igem.org/mediawiki/2019/5/5e/T--Technion-Israel--gel_3_9.jpeg )

    ***Figure 10:*** Gel electrophoresis of pBE-S + SP, 3.9.19

    + Nano drop results:

      |  | pBE-S |
      | --- | --- |
      | Concentration [ng/μl] | 343.8 |
      | 260/280 | 1.83 |
      | 260/230 | 1.59 |
      | Final Elution Volume [μl] | 35 |
    + Amplification of gBlocks– invertase from older closed pBE-S + SacC + Invertase – via PCR. The PCR products were cleaned from gel
    + Nano drop results:

      |  | Invertase |
      | --- | --- |
      | Concentration [ng/μl] | 422.6 |
      | 260/280 | 1.85 |
      | 260/230 | 2.13 |
      | Final Elution Volume [μl] | 35 |
    + ![]( https://static.igem.org/mediawiki/2019/b/bf/T--Technion-Israel--gel_3_9%281%29.jpeg )

      ***Figure 11:***  Gel electrophoresis of Invertase gBlock, 3.9.19
    + The Invertase and Glucose Oxidase (gBlock from 7/8) genes were cloned to pBE-S (containing HisTag). The products of reaction were transformed into *E. coli*
    + Measuring the activity of glucose oxidase and invertase in selected samples, in bacterial lysate and supernatant (pBE-S+SacC+Invertase 4 and pBE-S+SacB+Invertase 2 for invertase and pBE-S+SacC+Glucose Oxidase for glucose oxidase), in comparison to a calibration curve. No enzymatic activity was detected
    + Concentration of secreted enzymes from  **B. subtilis** supernatant (two strains: pBE-S+SacB+Invertase and pBE-S+AmyE+Glucose Oxidase) using amplicons, and purification using TCA and freeze-dry methods. The products were run on SDS-page, but unfortunately the results were not satisfactory
* + Meeting with Arnon Henn, who proposed to repeat the enzymes activity experiments in our project conditions (pH and bacterial content)
  + Discussion with Lena Daniell who corrected our Corrected our equations
* + Meeting with iGEMthon mentors
  + Meeting with Anat Sheer from Haifa municipality
  + Meeting with Mendi Rabinovitch, the Beit Biram High School managers

Week 19 (September 8-14)

* + Designing initial templates for our poster and presentation* + Colony PCR for *E. coli* Top 10 colonies using primers specific to the inserts. The gel had shown a lot of smears and no positive colony, maybe due to too long running time. We have repeated the experiment three more times, and got the same patterns in the gels, understanding that the problem might be in the gBlocks
    + Focusing on three main experiments and ordering the gBlocks needed. The final main experiments:
      1. Testing the inserts expression genes of GOx and Invertase inside the *Bacillus subtilis* (Lysates) and later on, testing the insert secretion under 174 different signal peptides, using Takara's library.
      2. Testing AmyE signal peptide as a secretion peptide of the inserts (supernatant and lysate)
      3. Testing the Honey Circuit secretion (supernatant and lysate)
    + Reverse PCR of pBE-S and pBE-S + AmyE signal peptide, Gel electrophoresis of PCR products and Clean-up of PCR products
    + Nano drop results:

      |  | pBE-S 1 | pBE-S 2 | pBE-S + AmyE 1 | pBE-S + AmyE 2 |
      | --- | --- | --- | --- | --- |
      | Concentration [ng/μl] | 37 | 76.7 | 101.5 | 102.2 |
      | 260/280 | 1.83 | 1.67 | 1.89 | 1.89 |
      | 260/230 | 0.21 | 0.1 | 0.55 | 0.3 |
      | Final Elution Volume [μl] | 30 | 30 | 30 | 30 |
    + Transformation of pBE-S + GOx and pBE-S + Invertase inserts and of pBE-S + AmyE + GOx or Invertase inserts, Gibson Assembly products into *E. coli* Top 10
    + Colony PCR for *E. coli* Top 10 colonies using primers specific to the inserts. The gel had shown a lot of smears and one colony was suspected as positive
    + ![]( https://static.igem.org/mediawiki/2019/5/5e/T--Technion-Israel--gel_11_9.jpeg )

      ***Figure 12:*** Colony PCR for *E. coli* top 10 colonies suspected as contain pBE-S + Invertase or pBE-S + AmyE, 13.9.19
    + Restriction enzymes (RE) reaction for validation of the insertion at pBE-S + GOx/Invertase and at pBE-S + AmyE + GOx+/Invertase, as a solution to the unclear smears that has been seen. Using EagI-Hf and PstI-HF RE which cut the plasmid out of the Invertase insert borders, and MluI-HF and PstI-HF RE which cut the plasmid out of the GOx insert borders. Gel electrophoresis of RE products
    + ![](https://static.igem.org/mediawiki/2019/4/4d/T--Technion-Israel--gel_13_9.png)

      ***Figure 13:*** Gel electrophoresis after restriction enzymes (RE) reaction for validation of the insertion at pBE-S + GOx/Invertase and at pBE-S + AmyE + GOx+/Invertase, 13.9.19
    + The colonies were sent for sequencing
    + Performing another TCA test for pBE-S + SacB Invertase, pBE-S + AmyE + GOx and W.T. afterwards we ran another SDS-PAGE for the new samples
    + ![]( https://static.igem.org/mediawiki/2019/0/06/T--Technion-Israel--SDS_11_9.png )

      ***Figure 14:*** SDS-PAGE after TCA concentration for pBE-S + SacB + Invertase , pBE-S + AmyE GOx and W.T.
    + Performing activity test invertase in decreasing pH levels
    + Performing activity test on glucose oxidase in decreasing pH levels
    + Performing test on bacterial resistance to hydrogen peroxide. The test has stopped due to human error* + Modelling the activity of glucose oxidase

Week 20 (September 15-23)

* + Single cut RE reaction for second validation of the insertion at pBE-S + GOx/Invertase and at pBE-S + AmyE + GOx+/Invertase, Using ClaI RE which cut in the middle of the Invertase insert, and XmaI RE which cut in the middle of the GOx insert. Gel electrophoresis of RE products
  + ![]( https://static.igem.org/mediawiki/2019/9/93/T--Technion-Israel--gel_15_9.png)

    ***Figure 15:***  Gel electrophoresis after single cut RE reaction of pBE-S + GOx/Invertase and at pBE-S + AmyE + GOx+/Invertase, Using ClaI RE (Invertase), and XmaI RE (GOx)
  + Transformation of pBE-S + GOx and pBE-S + Invertase inserts and of pBE-S + AmyE + GOx or Invertase inserts, Gibson Assembly products into *B. subtilis*
  + Transformation of The Honey Circuit into 5-alpha Competent *E. coli*
  + Colony PCR for *E. coli* Top 10 colonies using primers specific to the inserts
  + ![]( https://static.igem.org/mediawiki/2019/d/d8/T--Technion-Israel--gel_16_9.png)

    ***Figure 16:*** Colony PCR for 5-alpha component *E. coli* colonies suspected as contain pBE-S + Honey Circuit, 16.9.19
  + Glycerol stock for *E. coli* Top 10 + pBE-S + GOx/Invertase. Mini prep for pBE-S + GOx/Invertase
  + Nano drop results:

    |  | "Honey Circuit" |
    | --- | --- |
    | Concentration [ng/μl] | 570.5 |
    | 260/280 | 1.91 |
    | 260/230 | 2.21 |
    | Final Elution Volume [μl] | 40 |
  + Repeat Transformation of pBE-S + AmyE + GOx/Invertase into *E. coli* Top 10
  + Colony PCR for *E. coli* Top 10 colonies using primers specific to the inserts. The gel had shown a lot of smears and one colony suspected as positive
  + Single cut RE reaction for validation of the insertion of The Honey Circuit into the pBE-S plasmid, using XmaI RE which cut in the middle of the GOx insert. Another reaction with two RE for verification, using Spe-HF & XbaI RE which cut outside the insert borders. Gel electrophoresis of RE products
  + ![](https://static.igem.org/mediawiki/2019/f/f1/T--Technion-Israel--gel_17_9.png)

    ***Figure 17:***  Gel electrophoresis after single and double cut RE reaction of Honey Circuit, Using XmaI and Spe-HF&XbaI RE in accordance. 17.9.19
  + Glycerol stock for *E. coli* Top 10 + pBE-S + AmyE + GOx/Invertase. Mini prep for pBE-S + AmyE + GOx/Invertase
  + Nano drop results:

    |  | pBE-S + AmyE + GOx | pBE-S + AmyE + Invertase |
    | --- | --- | --- |
    | Concentration [ng/μl] | 500.6 | 367.1 |
    | 260/280 | 1.91 | 1.93 |
    | 260/230 | 2.19 | 2.18 |
    |
  + Colony PCR for *E. coli* Top 10 colonies of pBE-S + AmyE + GOx/Invertase, using primers specific to the inserts. The gel had shown a lot of smears and one colony suspected as positive
  + ![]( https://static.igem.org/mediawiki/2019/3/3a/T--Technion-Israel--gel_18_9.png )

    ***Figure 18:***  Colony PCR for *E. coli* Top 10 colonies suspected as contain pBE-S + AmyE + GOx/Invertase, 18.9.19
  + Transformation of pBE-S + AmyE + GOx/Invertase into *B. subtilis*
  + Glycerol stock for *B. subtilis* + pBE-S + AmyE + GOx/Invertase
  + ![]( https://static.igem.org/mediawiki/2019/e/e0/T--Technion-Israel--plates_19_9.jpeg)

    ***Figure 19:*** Plates with *B. subtilis* + AmyE + Invertase/GOx, Honey Circuit and WT to comparison (from up left, clockwise)' 19.9.19
  + His-tag purification of the samples pBE-S + AmyE + invertase and running in SDS-PAGE
  + ![]( https://static.igem.org/mediawiki/2019/e/eb/T--Technion-Israel--SDS_25_9.png)

    ***Figure 20:*** SDS PAGE of Invertase. Legend:
    L – Lysate,
    S – Supernatant,
    W – Wash,
    E – Elution. 23.9.19
  + His-tag purification of the samples pBE-S + AmyE + GOx and running in SDS-PAGE
  + ![]( https://static.igem.org/mediawiki/2019/8/81/T--Technion-Israel--SDS_25_9%281%29.png)

    ***Figure 21:*** SDS PAGE of GOx. Legend:
    L – Lysate,
    S – Supernatant,
    W – Wash,
    E – Elution. 23.9.19
  + Performing an enzymatic activity test on glucose oxidase in decreasing pH levels.
  + Performing catalase activity test in decreasing pH levels. The procedure has failed due to many bubbles created by the ammonium molybdate resulted in disrupted absorbance measurements
  + Performing test of bacterial resistance to hydrogen peroxide. Increased concentration of hydrogen peroxide prevented bacterial growth
  + Performing an activity test to pBE-S + AmyE + Glucose oxidase that was concentrated with the His-Tag method. Some activity has been detected* + Meeting with Faris Horani, who helped us understand how to model the pH influence on our system

Week 21 (September 24-28)

* + Transformation of pBE-S + GOx into *B. subtilis*
  + Glycerol stock for *B. subtilis* + pBE-S + AmyE + GOx/Invertase
  + Beginning wide library experiment, cutting pBE-S plasmid with MluI-HF and EagI-HF RE
  + ![]( https://static.igem.org/mediawiki/2019/2/28/T--Technion-Israel--gel_24_9.jpeg)

    ***Figure 22:*** Gel electrophoresis of cut pBE-S + Inevertase/GOx with positive control , 24.9.19
  + The PCR products were cleaned from gel
  + Nano drop results:

    |  | pBE-S + GOx | pBE-S + Invertase |
    | --- | --- | --- |
    | Concentration [ng/μl] | 53.9 | 80.5 |
    | 260/280 | 1.96 | 1.89 |
    | 260/230 | 0.77 | 0.39 |
    |
  + Infusion reaction has been performed (as described in "*B. subtilis* Secretory Protein Expression System (Library)" protocol)
  Transformation of pBE-S + Library + GOx/Invertase into *E. coli* HST08. Plating the transformants *E. coli* HST08 on large plates
* ![]( https://static.igem.org/mediawiki/2019/9/91/T--Technion-Israel--plates_24-9.jpeg)

  ***Figure 23:***  Plates with *E. coli* Stellar + library + Invertase(up) and *E. coli* Stellar + library + GOx, 24.9.19
* Resuspension of the colonies on the plate with LB. centrifugation and resuspension of all the pellets with the same volume of LB (5 ml). Mini prep for pBE-S + Library + GOx/Invertase
* Nano drop results:

  |  | pBE-S + Library + Invertase 1 | pBE-S + Library + Invertase 2 | pBE-S + Library + GOx 1 | pBE-S + Library + GOx 2 |
  | --- | --- | --- | --- | --- |
  | Concentration [ng/μl] | 495.95 | 555.4 | 605.4 | 525.1 |
  | 260/280 | 1.92 | 1.85 | 1.95 | 1.91 |
  | 260/230 | 1.72 | 1.23 | 2.10 | 1.90 |
  | Final Elution Volume [μl] | 60 | 60 | 60 | 60 |
* Transformation of pBE-S + Library + GOx/Invertase into *B. subtilis*. We have repeated the transformation to enlarge the colonies variation
* ![]( https://static.igem.org/mediawiki/2019/f/fe/T--Technion-Israel--plates_25_9.jpeg)

  ***Figure 24:*** Plates with *B. subtilis* + pBE-S + library + GOx/Invertase, 25.9.19
* ![]( https://static.igem.org/mediawiki/2019/d/dd/T--Technion-Israel--plates_27_9.png)

  ***Figure 25:*** Plates with *B. subtilis* + pBE-S + library + GOx/Invertase, 27.9.19
* Performing an activity test for glucose oxidase in The Honey Circuit. Very little activity has been detected
* Performing an activity test for AmyE invertase that was concentrated using the His-Tag. No activity has been detected
* Performing an activity test for Glucose oxidase AmyE+Sythetic circuit, activity has been detected
* Performing an enzymatic test for glucose oxidase +library. Some activity has been detected in the supernatent
* Performing an enzymatic test for Invertase + AmyE. No activity has been detected

- * Literature research regarding pH modeling in an enzymatic reaction- * The big iGEMthon event took place and was very successful
    * Going to a local excellence conference, arranged by Haifa Municipality in the Technion, and presenting our work to excellence coordinators from local school
    * Talking about our project in a lecture for new students in the Technion

Week 22 (September 29 – October 5)

* + Working on our presentation and poster for the Mini Jamboree next week* + Transformation of pBE-S + KatA promoter + mCherry into *E. coli*. Top 10 for validation of pKatA used in the "Honey Circuit"
    + ![]( https://static.igem.org/mediawiki/2019/8/88/T--Technion-Israel--plates_3_10.png)

      ***Figure 26:*** Plates of *E. coli* + pBE-S + pKatA + mCherry, 2.10.19
    + ![]( https://static.igem.org/mediawiki/2019/4/40/T--Technion-Israel--falcones_3_10.png)

      ***Figure 27:*** Falcones containing *E. coli* + pBE-S + pKatA + mCherry, red colored, 3.10.19
    + Mini prep for pBE-S + KatA promoter + mCherry
    + Nano drop results:

      |  | pBE-S + KatA promoter + mCherry |
      | --- | --- |
      | Concentration [ng/μl] | 211.2 |
      | 260/280 | 1.91 |
      | 260/230 | 2.20 |
    + Transformation of pBE-S + KatA promoter + mCherry into *B. subtilis*
    + Resuspension of the library colonies on the plate with LB, functions as starter
    + HIS-tag purification for the western blot, the samples we did are: "Honey Circuit", pBE-S + invertase and pBE-S + GOx, pBE-S + Library + GOx/Invertase and WT
    + Performing an enzymatic test for pBE-S + Library + Invertase, some activity has been measured
    + Performing an enzymatic test for pBE-S + Library + GOx, some activity was detected, however since we did not have fresh WT samples, we will have to repeat the test
    + Catalase pH test pre experiment section was performed. The procedure has failed due to high concentration of hydrogen peroxide
    + Catalase pH test pre experiment section was performed
    + Conducted an enzymatic test for catalase in decreasing pH levels
    + Performed an experiment – checking the validation and safety of "Bio-Castle" capsule which involved: bringing our bacteria to the log phase, syringing the bacteria into the capsule and incubation of the capsules in saline for 40 hours
    + Repeating the capsule experiment – due to bacterial contamination* + Further reasearch regarding pH modeling. Update of our equations

Week 23 (October 6-12)

* + Attended the Mini Jamboree in Tel Aviv University, in which we had fun meeeting the other Israeli teams and presenting our project* + Performing pKatA transcription assay as function of hydrogen peroxide concentration, twice, due to unclear results in the first try
    + First try of western blot for all the sample that went through His-tag purification, we only saw the positive control. We realized we need to repeat the test with larger volumes
    + ![](https://static.igem.org/mediawiki/2019/e/e8/T--Technion-Israel--WB_6_10.png)

      ***Figure 28:*** Western blot of all the samples, 7.10.19
    + Repeat all the samples again with His-tag purification, this time we grew the bacteria in 500 ml LB
    + Conducted catalase pH experiment
    + Performing enzymatic test on pBE-S + Library + GOx, no activity was detected
    + Performing enzymatic test on pBE-S + Library + Invertase, some activity was detected* + Building of the stocchastic model
      + Modeling the activity of catalase

Week 24 (October 13-19)

* + "Honey Circuit" validation – the experiment contains 5 times intervals and 3 samples, 2 controls
  + Demonstration experiments – validating our system – by showing the way every part work separately
  + Catalase activity test to test the capsules
  + Activity test for the products tested in the western blot* + Implementation of pH change in our equations based on GDL concentrations

![](https://static.igem.org/mediawiki/2019/c/c6/T--Technion-Israel--Sponsors-Bioanal.png)

![](https://static.igem.org/mediawiki/2019/8/85/T--Technion-Israel--Sponsors-BioCastle.png)

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My First Website

![](https://static.igem.org/mediawiki/2017/a/ab/T--TECHNION-ISRAEL--pin.png)

Department of Biotechnology & Food Engineering

Technion – Israel Institute of Technology

Haifa 32000, Israel

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igem.technion.2019@gmail.com


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# Team:Technion-Israel/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Technion-Israel/Results

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* [introduction](#m1)
* [Secretion](#m2)
* [Enzymatic Activity Test](#m3)

# Introduction

Our goal is to achieve the same composition of sugars as in bee-made honey, which will turn to the familiar substance called "honey" as soon as the solution will be dried, just like the bees do it.

We aimed to design a **system** that will monitor the solution conditions and **regulate** the production of its contents accordingly.

The chosen bacteria for that mission was ***Bacillus subtilis* RIK1285**, a bacterium known to effectively secrete proteins, while *E. coli* (Top 10/ 5 alpha/ stellar) used as a tool for the cloning and construction of our recombinant enzymes.

To obtain our self-regulated system, we decided to use synthetic biology tools to create our special **"Honey Circuit"**, described in detail in the "design" chapter. For the fabrication of this circuit, we used a "pBE-S" plasmid backbone, which is a *B. subtilis/ E. coli* shuttle vector provided by the TaKaRa company.

The Honey Circuit we created was cloned into pBE-S backbone ligated with a signal peptide at the "aprE SP" area and inserted with gBlock at the "Multi cloning site (MCS)" area (Figure 1). The rest of the clones were created similarly.

![Genetic circuit](https://static.igem.org/mediawiki/2019/6/63/T--Technion-Israel--hp--takara.png)

**Figure 1:** Map of pBE-S vector plasmid from TaKaRa

(2018. *B. subtilis* Secretory Protein Expression System. TaKaRa Bio inc.)

Our target proteins are [Glucose Oxidase (GOx)](https://2019.igem.org/Team%3ATechnion-Israel/Design#gox) and [invertase](https://2019.igem.org/Team%3ATechnion-Israel/Design#inv), enzymes that degrade **glucose** and **sucrose**, respectively, and are key enzymes in the creation of honey inside the bee's stomach.

Initially, we have chosen two known *B. subtilis* signal peptides, **AmyE** and **SacB**, as candidates for the secretion of our target proteins. The signal peptides employed to enable the secretion of the translated proteins which are genetically located right after them, downstream in the plasmid.

After successful insertion of the signal peptides and the proteins into the backbone by ligation and Gibson assembly, we have examined four approaches to concentrate the proteins in the supernatant and lysate: **His-Tag column, TCA, Freeze-drying and Amicon Ultra-15 Centrifugal Filter** . The concentration process was essential to enable us to see the proteins in the SDS-PAGE assay (30 micrograms of protein per ml are required) and determine the presence of the desired enzymes, either in the bacterial lysate or supernatant.

After additional optimization of the gBlocks, we reordered them as new sequences, not containing a stop codon between the enzyme gene and the Histag following it, as it is normally found in the pBE-S backbone. By using a **Histag column**, we minimized our efforts concentrating the proteins and separated them more specifically from the rest of the proteins in the supernatant or lysate.

To further confirm the presence of GOx and invertase in the solution and to determine whether they are active, we conducted **protein activity assays**.

# Secretion

### Introduction

The goals of the following experiments were to confirm the **expression** and **secretion** of our target enzymes. In order to determine the location of the enzymes, both the bacterial **lysate** and the **supernatant** were tested. First, the samples were **concentrated** via several methods. Then, **SDS-PAGE** and **Western blot** methods were employed in order to confirm the presence of the enzymes. Finally, **activity tests** have been conducted.

### Method

![Genetic circuit](https://static.igem.org/mediawiki/2019/5/5d/T--Technion-Israel--SchemeHashtanot.png)

**Figure 2:** Experimental scheme

Bacteria were grown in LB and separated into lysate and supernatant.

The proteins were concentrated using the following methods: TCA, Freeze-Drying, Amicon Ultra-15 Centrifugal Filter and His-Tag column.

**To check our results, we used SDS-PAGE, Western blot and activity tests**.

As part of our project, we inserted several combinations of signal peptides and our two proteins, [Glucose oxidase (GOx)](https://2019.igem.org/Team%3ATechnion-Israel/Design#gox) and [invertase,](https://2019.igem.org/Team%3ATechnion-Israel/Design#inv) into our model bacteria. Then we tested the protein expression and secretion.

Each test was divided into these subgroups:

![TAG](https://static.igem.org/mediawiki/2019/c/cf/T--Technion-Israel--table1aresultsHASTANOT.png)

**Table 1:**  The Bacteria samples tested

1. **AmyE signal peptide** – Testing both the bacteria lysate (intercellular medium) and supernatant (outer cell fluid that held the secreted proteins).

2. **Library** – A collection of 173 different signal peptides, that were inserted simultaneously into the bacteria. Assuming at least one signal peptide is successful. Testing both the bacteria lysate and supernatant.

3. **SacB signal peptide** - Testing both the bacteria lysate and supernatant.

4. **Honey Circuit** – Our BeeFree synthetic circuit with the AmyE signal peptide and GOx enzyme. Testing both the bacteria lysate and supernatant.

5. **No Signal Peptide (SP)** – Bacteria with the plasmid with no signal peptide, testing only the bacteria lysate.

In addition, we used a **Wild type** of *B. subtilis* as a **control** native bacterium.

### Results

Here, we present the progress we have made to identify the proteins for our BeeFree system using the different methods mentioned above.

![TAG](https://static.igem.org/mediawiki/2019/9/96/T--Technion-Israel--Fig_6.png)

**Figure 3:** SDS-PAGE test results using His-Tag concentration for **Invertase with AmyE signal-peptide**

As can be seen in Figure 3, our invertase His-Tag concentration did not result in any visible band in the expected area of invertase.

![TAG](https://static.igem.org/mediawiki/2019/6/6e/T--Technion-Israel--Fig_4.png)

**Figure 4:** SDS-PAGE test results using His-Tag concentration for **GOx with AmyE signal-peptide**

![TAG](https://static.igem.org/mediawiki/2019/7/7b/T--Technion-Israel--FIG5resultsHASTANOT.png)

**Figure 5:** SDS-PAGE test results using His-Tag concentration for **GOx without signal-peptide**

![TAG](https://static.igem.org/mediawiki/2019/b/b8/T--Technion-Israel--FIG6SynWithWTresultsHASTANOT.png)

**Figure 6:** SDS-PAGE test results for the **Honey circuit** by His-Tag concentration method

As can be seen in figures 4-6, there is a weak band corresponding to the size of the standard GOx. However, it was difficult to reach a concrete conclusion from looking at the gels presented in figures 4-5, since we had encountered a problem with our WT samples preventing us from running them in the gel. Luckily, the WT supernatant sample did run, as can be seen in figure 6, and no band was observed.

The result for the WT supernatant was encouraging since we have detected the protein in its expected size in the "Honey Circuit" supernatant, and not in the WT sample.

Moreover, there was a strong band in the range of 40-48 kDa in the sample containing the AmyE signal peptide (Fig. 4,6), as can also be seen in the Western Blot experiment, about which we will elaborate below.

In summary, we got inconclusive results from our SDS-PAGE runs. The gels seen in figures 4-5 gave us a vague assumption of the expression and the secretion of GOx since we couldn’t compare it to the WT. The "Honey Circuit" results (figure 6) were more positive, as we **detected a protein in the expected size**, and could compare it to the WT.
Encountering time limitations, we decided not to repeat those tests but to move on to a more specific test- the Western Blot.

![TAG](https://static.igem.org/mediawiki/2019/1/12/T--Technion-Israel--table2aresultsHASTANOT.png)

**Table 2:** Summary of the SDS-PAGE results

### Western Blot

Western blot (WB) is a **more precise** method compared to SDS-PAGE. By using antibodies to detect our protein's His-tag, it can be guaranteed that our protein had been expressed and secreted by the bacteria.

![TAG](https://static.igem.org/mediawiki/2019/8/89/T--Technion-Israel--Figure_7_Western_Blot.png)

**Figure 7: WB** test results for the **Honey circuit** by His-Tag concentration method

As seen in Figure 7, the WB test yield a **definite result** in the Honey circuit strain's supernatant, meaning that our bacteria **express** and **secrete** the GOx protein.

However, the results are somewhat unexpected. Firstly, the size of the protein was different than the expected size, which according to the standard and other studies was is in the range of 67-80 kDa.

Nevertheless, we could not find a reliable source explaining the different size of our GOx. Our hypothesis is that the signal peptide affects the translation of the enzyme in the bacteria, resulting in a different structure of the protein monomer. Another assumption we have is that a part of the protein stays in the membrane during the secretion process as part of the secretion pathway, as described in the "description" chapter, which affects the protein size.

Secondly, The Honey Circuit contains the AmyE signal peptide, yet the sample containing AmyE+GOx was not visible in the WB. The solution to that may be the difference between the promoters of each strain, the Honey Circuit strain promoter is pLac and the AmyE GOx strain promoter is pAprE.

Nonetheless, the experiment ought to be repeated in order to get a conclusive result and to optimize our system.

# Enzymatic Activity Test

We have performed plenty of assays to confirm that our inserted genes lead to the production and secretion of our target proteins. However, we also need to verify that our enzymes **remain active**. Therefore, we had performed several assays that verified and quantified the enzymatic activity of the three enzymes created by the recombinant *Bacillus subtilis*:
[invertase](https://2019.igem.org/Team%3ATechnion-Israel/Design#inv), [glucose oxidase](https://2019.igem.org/Team%3ATechnion-Israel/Design#gox) and [catalase.](https://2019.igem.org/Team%3ATechnion-Israel/Design#cat)

### Invertase Assay

**Invertase** is an enzyme that cleaves sucrose to form glucose and fructose. Thus, a precise method that will detect either glucose or fructose can indicate the amount of sucrose cleaved by the enzyme. The test we used is based on the **"reducing sugars" assay**, in which reducing sugars react with the **yellow picric acid** to form picremic acid, which is red (Figure 8). Therefore, the **absorbance** of red light (492nm) could indicate the amount of sucrose cleaved, which indicates the **enzymatic activity**.

![TAG](https://static.igem.org/mediawiki/2019/1/1e/T--Technion-Israel--hp--PicricAcid.jpg)

**Figure 8:** Illustration of the reaction of picric acid with reducing sugars

To verify that the **activity-absorbance correlation** remains linear under the experiment's conditions, we performed the experiment using a commercial enzyme, creating a **calibration curve**. We used increasing concentrations of commercial invertase (with a well-determined specific activity) and a constant saturated concentration of sucrose. Then, we checked the 492 nm light absorbance and plotted the **calibration curve** (Figure 9).

![TAG](https://static.igem.org/mediawiki/2019/1/11/T--Technion-Israel--Lior-Fig_9.png)

**Figure 9:** (A) Calibration curve for invertase activity – change in absorbance at 492 nm for different activity values. (B) The calibration curve tubes

After the cloning process had been completed, we performed an activity test on the **bacterial invertase**. We tested both **bacterial lysate** and **bacterial supernatant** to determine whether active invertase had been produced and secreted by evaluating the activity in each sample. The recombinant bacteria samples were **compared to wild type (WT)** samples.

The first colony tested contained the invertase enzyme with AmyE as a signal peptide. Unfortunately, no notable activity was detected (Figure 10).

These results, in addition to the results observed in the SDS-PAGE experiment, indicating that the AmyE signal peptide may interfere with either the enzyme's production or its activity.

![TAG](https://static.igem.org/mediawiki/2019/c/c7/T--Technion-Israel--Lior-Fig_10.png)

**Figure 10:** The activity of invertase and AmyE in different samples, indicated by the absorbance at 492 nm

In addition to evaluating the **activity of invertase with AmyE**, we used a library of signal peptides aiming to obtain enzymatic activity for the bacterial invertase (FigureX4). The Bacterial **supernatant** activity detected is **0.18U/ml** and the activity detected in the **lysate** solution is 0.12U/ml. The activity detected for the **WT** sample is observed to be **significantly lower** in comparison to the bacterial samples, both in the supernatant and the lysate, i.e, one or more of the library signal peptides has enabled both secretion and activity of the enzyme.

![TAG](https://static.igem.org/mediawiki/2019/f/fd/T--Technion-Israel--Lior-Fig_11.png)

**Figure 11:** The activity of invertase and Library in different samples, indicated by the absorbance at 492 nm

### Glucose Oxidase Assay

[Glucose Oxidase (GOx)](https://2019.igem.org/Team%3ATechnion-Israel/Design#gox) catalyzes the oxidation of glucose to D-gluconic-lactone and hydrogen peroxide. Therefore, a precise method to determine the amount of hydrogen peroxide found in the solution could indicate the activity of the glucose oxidase. In this experiment, we used the assistance of another commercial enzyme- **horseradish peroxidase (HRP)** and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Using **hydrogen peroxide**, the HRP **catalyzes the oxidation** of reduced ABTS, which is colorless, to oxidized ABTS, which is turquoise. While using excess ABTS and HRP, the absorbance of 416 nm light could indicate to us the amount of hydrogen peroxide created by the glucose oxidase, and therefore - the glucose oxidase activity.

The test is based on the following reactions:

![invertase](https://static.igem.org/mediawiki/2019/e/e1/T--Technion-Israel--hp--Nuscha.png)

To verify that the absorbance-activity relation remains linear under the experiment's conditions, we performed the experiment using commercial GOx with increasing activity, measured the 416 nm light absorbance and plotted a graph to demonstrate that relation. Fortunately, we have proved the **absorbance-activity relation** linearity under the experiment's conditions (Figure 12).

![TAG](https://static.igem.org/mediawiki/2019/d/dd/T--Technion-Israel--Lior-Fig_12.png)

**Figure 12:** Calibration curve for glucose oxidase activity – change in absorbance at 416 nm in different activity values

After the cloning process had been completed, we performed the activity test on both the supernatant and the lysate of genetically engineered bacteria as well as WT bacteria as control. The first colony tested contained a gene for **glucose oxidase** and the **AmyE signal peptide**. The absorbance detected in the recombinant Bacteria was significantly higher, in both **supernatant** and **lysate** (Figure 13). The evaluated activity of glucose oxidase is **0.46U/ml** in the bacterial **supernatant** and **0.45U/ml** in the **lysate** solution.

![TAG](https://static.igem.org/mediawiki/2019/3/39/T--Technion-Israel--Lior-Fig_13_GOx-AmyE.png)

**Figure 13:** The activity of glucose oxidase and AmyE in different samples, indicated by the absorbance at 416 nm

Aiming to obtain increased activity, we used a **library of signal peptides** for the glucose oxidase insert, similar to the experiment performed on invertase. Unfortunately, we could not detect any notable activity (Figure 14).

![TAG](https://static.igem.org/mediawiki/2019/f/f6/T--Technion-Israel--Lior-Fig_14_GOx-Library.png)

**Figure 14:** The activity of glucose Oxidase and Library in different samples, indicated by the absorbance at 416 nm

### Catalase Assay

The **catalase** enzyme decomposes hydrogen peroxide to water and molecular oxygen. Since water cannot function as an **indicator** in aqueous solution and molecular oxygen tends to evaporate quickly, the main indicator for catalase activity had to be the hydrogen peroxide. Hence, we had performed an experiment that yielded us the **hydrogen peroxide-absorbance relation**, and therefore the catalase activity-absorbance relation. We had decomposed hydrogen peroxide using catalase, and afterward added the hydrogen peroxide-catalase solution to a solution containing **ABTS** and **HRP enzyme**. The blank (negative control) contained the initial amount of hydrogen peroxide and no enzyme. Each sample's absorbance was subtracted from the blank sample's absorbance to evaluate the amount of hydrogen peroxide decomposed by the catalase.

The reaction on which the test is based on is:

![invertase](https://static.igem.org/mediawiki/2019/b/ba/T--Technion-Israel--hp--Nuscha2.png)

To validate this method for testing, we had to verify the relation between the amount of hydrogen peroxide and the 416 nm light absorbance measured is linear. To do so, we have tested the reaction of constant amounts of ABTS and HRP to increasing amounts of hydrogen peroxide. After performing the test and plotting the hydrogen peroxide concentration-absorbance graph, we have deduced that this method is in fact, reliable (Figure 15).

![TAG](https://static.igem.org/mediawiki/2019/2/22/T--Technion-Israel--Lior-Fig_15_Catalase-Verfying_Graph.png)

**Figure 15:** Verification of Linear Ratio between Hydrogen Peroxide and Absorbance

The catalase enzyme is **naturally produced** and **secreted** by the ***Bacillus Subtilis*** Bacteria, thus, a comparison between WT and genetically engineered bacteria is not needed for that enzyme. We had plotted a calibration curve using increasing amounts of commercial catalase enzyme and used it to determine the activity of the bacterial lysate and supernatant. From that experiment, we had evaluated the bacterial **lysate** activity to be **22.26 U/ml** and the bacterial **supernatant** activity to be **12.26U/ml**. These results **confirm that the bacteria produce the catalase and partially secretes the enzyme.**

![TAG](https://static.igem.org/mediawiki/2019/7/73/T--Technion-Israel--Lior-Fig16-catalase_activity.png)

**Figure 16:** The activity of glucose Oxidase and Library in different samples, indicated by the absorbance at 416 nm

### Conclusions

![TAG](https://static.igem.org/mediawiki/2019/6/6d/T--Technion-Israel--table3resultsHASTANOT.png)

**Table 3:** Summary of secretion and activity assays

As presented in Table 3, the **Honey Circuit supernatant is the most successful strain** (as seen in the [demonstration](https://2019.igem.org/Team%3ATechnion-Israel/Demonstrate#t3)). We have achieved positive results in all categories, SDS-PAGE, Western Blot and activity tests, for that sample. The fact that we saw the secretion and activity of GOx is encouraging as it serves as the proof of concept for the project. Despite the success we had with GOx, not all of the secretion tests turned out to be successful. Due to time limits, we have decided to continue only with the AmyE signal peptide.

We are aiming to continue and research the **SacB signal peptide** as well as the commercial library of 173 signal peptides to find the **most suitable signal peptides** for our system. Moreover, tests for unexamined samples should be performed.

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Department of Biotechnology & Food Engineering

Technion – Israel Institute of Technology

Haifa 32000, Israel

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