Team:Technion-Israel/Notebook

Basic Bootstrap Template

- 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

- 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

- 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

- 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

- 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
Figure 1: Refractometer and commercial honey tested, 6.6.19
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

- 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

- 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

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

- Ligation to pBE-S with different signal peptides: AmyE, SacB, SacC
- Transformation to E. coli Top 10
- Figure 3: E. coli Top 10 + pBE-S + Signal Peptides(SP), rest, 4.7.19

- Colony PCR for the E. coli Top 10 colonies using insert specific primers for the three signal peptides
- Figure 4: Gel electrophoresis of pBE-S + SP, 7.7.19
- Mini prep of pBE-S + Signal peptides and sequencing

Reverse PCR of pBE-S + Signal peptides
Gel electrophoresis of PCR products
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

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

- 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
- 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

- 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

- Designing group T-shirts
- Initial wiki graphics
- Colony PCR for E. coli Top 10 colonies using primers specific to the inserts
- 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
- 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

- 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

- 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

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
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

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

- 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
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
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
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

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
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
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
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
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
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
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
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

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
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)

E. coli

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
Figure 24: Plates with B. subtilis + pBE-S + library + GOx/Invertase, 25.9.19
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

- 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"
- Figure 26: Plates of E. coli + pBE-S + pKatA + mCherry, 2.10.19
- 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

- 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
- 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

- "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

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