iGEM 2021 — Team Saint_Joseph

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Saint Joseph/Notebook

![](https://static.igem.org/mediawiki/2021/4/4c/T--Saint_Joseph--logo-200.png)

[Project](/Team%3ASaint_Joseph)

[Description](/Team%3ASaint_Joseph/Description)
[Proposed Implementation](/Team%3ASaint_Joseph/Implementation)
[Contribution](/Team%3ASaint_Joseph/Contribution)
[Wiki Design](/Team%3ASaint_Joseph/Wiki)

[Human Practices](/Team%3ASaint_Joseph/Human_Practices)

[Education & Communication](/Team%3ASaint_Joseph/Communication)
[Inclusion](/Team%3ASaint_Joseph/Inclusivity)
[Education](/Team%3ASaint_Joseph/Education)
[SDG Impact](/Team%3ASaint_Joseph/Sustainable)
[Source Of Inspiration](/Team%3ASaint_Joseph/Source_Of_Inspiration)

[Engineering](/Team%3ASaint_Joseph/Engineering)

[Experiments](/Team%3ASaint_Joseph/Experiments)
[Results](/Team%3ASaint_Joseph/Results)
[Notebook](/Team%3ASaint_Joseph/Notebook)

[Team](/Team%3ASaint_Joseph/Team)

[Members](/Team%3ASaint_Joseph/Team)
[Attributions](/Team%3ASaint_Joseph/Attributions)
[Collaborations](/Team%3ASaint_Joseph/Collaborations)

[Parts](/Team%3ASaint_Joseph/Parts)

![]( 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gAgggASCACAAQRAJBABAAIIgAggQgBE8W6QUrXJoopEKEOS5e0t3ju0ggW3W13Yd92P1MSgAh1WHnDUrqijZ533Z03U9i0+zfW3BruCnwmAxHqwOnH/S2uFdojetyNX7uNwoaXlFwhRG0FuXw9IkJdIEIDeNHcVhrPj77XT1Hx2A83+Y5Y/BkGH8Bq2I2ACA3gqPDszx5sKgO/jxvm3XeFnxZ5cOr266eb7xu/7uczJB2shu2T/S+8Qk89c/DCwsAMC8Apx8C31ypraLw/o788KBcGrl4K0lsGnjdtRpk0Xg0bIrSIt6o0N3zdbsvk3TpKx32T4I458YF717Dvf9AuK1ssoMFF9K4RLLtAID4sWHiCNEVvEUrFtwjEBuvzX9f2eOo+ajRNIC5ofSz1FsGFCPHBgghtc16KMEvAfNziJGmM1iJY1/fPkqV3TwJ88gnlSWP0P7NcLI4QMJzS86JTK5DGaC9C5QQM0iOTKVKWNMeMuUalEpZrM5eCdV3/BGmOGSKcp72EqGAmbsmI1NYIEWTR7Ja2ETAMd8Lq6c+SARgzDVt+oZaVJ2AKs3TONabzMut6hI+LfOVLgYD+iAiu+0hRNUaJUEmRWAbUCzpTKo2YkhJ5aH09Qj3cf4730QJ7nIB+uKV9QoIMGYaRl2rK4bgiIoN2iGFuEyVgjIwIHu7b4w6V7Jx46BBQC6dDy/qHyVCMvnjfulYUY3YpzeGYgCoKlUswh8lgjI4I1bgz4xmy7CFCdIgOPuN/noblIIbhxEYEDwgRBVaWzhVHTBoebUbsRPAojywt2CwepcTTbgKd4bp5sU3ydJc4RID5xFaEatyT4ynxSXvJWnALuRwpSt3iOeSohytG4yx7mkrF98XjPBUpH8fGX83/AYZVqjkejyobAAAAAElFTkSuQmCC)

# Loading

[Open Notebook](https://static.igem.org/mediawiki/2021/d/d1/T--Saint_Joseph--Notebook.pdf)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Saint Joseph/Engineering

![](https://static.igem.org/mediawiki/2021/4/4c/T--Saint_Joseph--logo-200.png)

[Project](/Team%3ASaint_Joseph)

[Description](/Team%3ASaint_Joseph/Description)
[Proposed Implementation](/Team%3ASaint_Joseph/Implementation)
[Contribution](/Team%3ASaint_Joseph/Contribution)
[Wiki Design](/Team%3ASaint_Joseph/Wiki)

[Human Practices](/Team%3ASaint_Joseph/Human_Practices)

[Education & Communication](/Team%3ASaint_Joseph/Communication)
[Inclusion](/Team%3ASaint_Joseph/Inclusivity)
[Education](/Team%3ASaint_Joseph/Education)
[SDG Impact](/Team%3ASaint_Joseph/Sustainable)
[Source Of Inspiration](/Team%3ASaint_Joseph/Source_Of_Inspiration)

[Engineering](/Team%3ASaint_Joseph/Engineering)

[Experiments](/Team%3ASaint_Joseph/Experiments)
[Results](/Team%3ASaint_Joseph/Results)
[Notebook](/Team%3ASaint_Joseph/Notebook)

[Team](/Team%3ASaint_Joseph/Team)

[Members](/Team%3ASaint_Joseph/Team)
[Attributions](/Team%3ASaint_Joseph/Attributions)
[Collaborations](/Team%3ASaint_Joseph/Collaborations)

[Parts](/Team%3ASaint_Joseph/Parts)

![]( 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gAgggASCACAAQRAJBABAAIIgAggQgBE8W6QUrXJoopEKEOS5e0t3ju0ggW3W13Yd92P1MSgAh1WHnDUrqijZ533Z03U9i0+zfW3BruCnwmAxHqwOnH/S2uFdojetyNX7uNwoaXlFwhRG0FuXw9IkJdIEIDeNHcVhrPj77XT1Hx2A83+Y5Y/BkGH8Bq2I2ACA3gqPDszx5sKgO/jxvm3XeFnxZ5cOr266eb7xu/7uczJB2shu2T/S+8Qk89c/DCwsAMC8Apx8C31ypraLw/o788KBcGrl4K0lsGnjdtRpk0Xg0bIrSIt6o0N3zdbsvk3TpKx32T4I458YF717Dvf9AuK1ssoMFF9K4RLLtAID4sWHiCNEVvEUrFtwjEBuvzX9f2eOo+ajRNIC5ofSz1FsGFCPHBgghtc16KMEvAfNziJGmM1iJY1/fPkqV3TwJ88gnlSWP0P7NcLI4QMJzS86JTK5DGaC9C5QQM0iOTKVKWNMeMuUalEpZrM5eCdV3/BGmOGSKcp72EqGAmbsmI1NYIEWTR7Ja2ETAMd8Lq6c+SARgzDVt+oZaVJ2AKs3TONabzMut6hI+LfOVLgYD+iAiu+0hRNUaJUEmRWAbUCzpTKo2YkhJ5aH09Qj3cf4730QJ7nIB+uKV9QoIMGYaRl2rK4bgiIoN2iGFuEyVgjIwIHu7b4w6V7Jx46BBQC6dDy/qHyVCMvnjfulYUY3YpzeGYgCoKlUswh8lgjI4I1bgz4xmy7CFCdIgOPuN/noblIIbhxEYEDwgRBVaWzhVHTBoebUbsRPAojywt2CwepcTTbgKd4bp5sU3ydJc4RID5xFaEatyT4ynxSXvJWnALuRwpSt3iOeSohytG4yx7mkrF98XjPBUpH8fGX83/AYZVqjkejyobAAAAAElFTkSuQmCC)

# Loading

# Engineering Success

![](https://static.igem.org/mediawiki/2021/2/2a/T--Saint_Joseph--Engineering-Jambatron-bg.png)
![](https://static.igem.org/mediawiki/2021/2/2a/T--Saint_Joseph--Engineering-Jambatron-btm.png)

![Learn Design Build Test, are aligned as a circle](https://static.igem.org/mediawiki/2021/2/23/T--Saint_Joseph--Cycle-Learn.png)
![](https://static.igem.org/mediawiki/2021/c/cb/T--Saint_Joseph--Cycle-Test-1.png)
![](https://static.igem.org/mediawiki/2021/3/34/T--Saint_Joseph--Cycle-Test-2.png)
![](https://static.igem.org/mediawiki/2021/3/3a/T--Saint_Joseph--Cycle-Design.png)
![](https://static.igem.org/mediawiki/2021/b/ba/T--Saint_Joseph--Cycle-Build.png)

# Learn

We started our project by making research on obtaining statistical data on paper towel
usage, in addition to searching for specific organisms that will increase the productivity of our
selected enzyme, cellulase. After finding about CelAB in *T. turnerae* (who is in a symbiotic
relationship with *Lyrodus pedicellatus* a.k.a. shipworm) and EGII in *T. reesei*, the
design section of
our project was initialized.

![](https://static.igem.org/mediawiki/2021/5/56/T--Saint_Joseph--Engineering-divider-2.png)

# Design

Recognizing the importance of the design section, we created guidelines to reach our
goals regarding the other stages of the engineering process. We worked on the processes that would
need to be implemented and learned about the computational/laboratory tool and techniques to be used
in the making of our enzyme. We took the catalytic region of CelAB and added a 6XHis tag at the end
to harvest our proteins way more easily. Our goal was to isolate the enzyme of the bacteria and use
it as a standalone material. Since the cellulase does hydrolysis, we only needed water and
substrates for it to work.

Codon Optimized CelAB and a His Tag

As for the EGII we removed the linker region, CBM domain and the signal
peptide from the original sequence. We codon-optimized (for use in DH5α) the remaining sequence and
again we added a 6XHis Tag at the end for the same reason. Later on, we did two mutations of our
EGII C99V and D185S.

![A Figure explaining removed Signal Peptide, CBM One, Linkers from Endo glucanase 2. Codon optimized the clipped sequence. Mutated the optimized sequence thus remaining C99V and D185S](https://static.igem.org/mediawiki/2021/6/6b/T--Saint_Joseph--Diagram-DNA.png)

![](https://static.igem.org/mediawiki/2021/2/2c/T--Saint_Joseph--Engineering-divider-3.png)

# Build

Furthermore, we inscribed our designed sequence to E.
Coli. (We inserted the cloning vector into D185⍺ and the expression vector into BL21.) We used
laboratory techniques such as PCR, gel electrophoresis, gene transferring etc. We used pET29b+
vector and inserted the CelAB, EGII, EGII (C99V), and EGII(D185S) parts to the cutting region from
NdeI to XhoI.

![A Figure showing steps: 1. Literatture Review, 2. Bioinformatic analysis                and design, 4. Restriction Digestion, 5. Recovery From Agarose Gel, 6. Ligation, 7. Compotent cell preperation, 8. Selective Media, 9. IPTG Induction, 10. Protein isolation, 11. Protein concentration analysis                with Bradford Assay, 12. Determination of enzyme activity](https://static.igem.org/mediawiki/2021/a/a2/T--Saint_Joseph--Diagram-Build.png)

![](https://static.igem.org/mediawiki/2021/c/ca/T--Saint_Joseph--Engineering-divider-4.png)

# Test

To see if the expression of our enzymes was successful,
we conducted a Bradford Analysis. If the indicator turned blue, it meant that the isolation
experiment was successful. All of them except EGII D18S turned blue As a result, we reached the
conclusion that D185S was a lethal mutation for the bacteria.

![](https://static.igem.org/mediawiki/2021/6/69/T--Saint_Joseph--Bradford-Assay.jpg)

| Enzyme | Absorbance | Concentration (mg/mL) |
| --- | --- | --- |
| CelAB | 0.3726625 | 0.127356103179867 |
| EGII | 0.2968375 | 0.3006875 |
| EGII (C99V) | 0.100889210792698 | 0.102233062236029 |

To see the activity of enzymes, we did a CMCase activity analysis. We made graphs
according to the absorbance and concentration of our enzymes. We calculated enzyme activities
according to our reaction time. CelAB got 17,5 U/min, EGII got 24,85 U/min and EGII(C99V) got 19,26
U/min.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Saint Joseph/Experiments

![]( 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)

# Loading

# Experiments

![](https://static.igem.org/mediawiki/2021/2/2a/T--Saint_Joseph--Engineering-Jambatron-bg.png)
![](https://static.igem.org/mediawiki/2021/2/2a/T--Saint_Joseph--Engineering-Jambatron-btm.png)

# LB Broth Media Preparation

Growth Medium Preparations
Luria-Bertani (LB) medium is used for the establishment of cell cultures in all experiments to be
carried out.

* **Preparation of LB Broth (Liquid) Media**
  1. 25 grams of LB Broth Powder (Tripton, Yeast Extract, NaCl 2:1:2) poured into a bottle
     with a sterile screw capped bottle.
  2. Complete the volume with 1 liter of ddH2O. Cover it halfway and wrap it with foil
     autoclave tape.
  3. Autoclave at 121°C for 15 minutes.
  4. After cooling at room temperature, store at 4°C.
* **Preparation of LB Agar (Solid) Medium**
  1. Prepare 1 Liter of LB broth in a 2-liter flask using the first two steps described
     above. Add 15 grams of bacterial agar (1.5%).
  2. Autoclave at 121 °C for 15 minutes.
  3. Cool down to approximately 50 °C.
  4. If antibiotics are to be added, they must be added at this stage with the right
     temperature, otherwise the antibiotic added to a warm environment will degrade
     rapidly. Mix by vigorously rotating the bottle.
  5. The medium is poured into petri dishes. When pouring the petri dish, be careful not to
     drop the temperature too much.
  6. Approximately 25-30 ml of LBA can be poured into a petri dish.

![](https://static.igem.org/mediawiki/2021/5/56/T--Saint_Joseph--Engineering-divider-2.png)

We started the transformation experiments by preparing competent cells according to
the protocol. We used *E. coli* DH5a bacteria. We inserted plasmid DNA to these bacteria by heat-shock
transformation.

# Competent Cell Recovery and Transformation

* We ordered our plasmids from IDT
  ![](https://static.igem.org/mediawiki/2021/c/ca/T--Saint_Joseph--plasmids-puc.png)
* **Making *E. coli* DH5α strain competent (with CaCl2 Method)**
  A single colony is taken from the *E. coli* DH5α cultured solid medium and inoculated into 10 ml
  of LB medium. The culture is incubated at 37°C overnight. Then, overnight culture is incubated
  on ice for 10 minutes. It is centrifuged at 14000 rpm at 4°C for 5 minutes in a refrigerated
  centrifuge and the supernatant is discarded. 2 ml of 0.1 M CaCl2 added to the pellet and keeped
  on ice for 30 min. It is centrifuged at 14.000 rpm at 4°C for 5 minutes in a refrigerated
  centrifuge and the supernatant is discarded. 0.4 ml of 0.1 M CaCl2 added to the pellet and
  stored at 4oC (it must not be kept no longer than 18 hours.) Each transformations made by 200
  microliters of these competent cells bu using heat shock.
* **Plasmid isolation (For stockpiling, with strain DH5α)**
  The plasmid isolation is done with the Thermofisher GeneJet Plasmid Miniprep kit, according to
  the protocol recommended by the manufacturer.

  ![Pellet bacteria by                               centrifugation, Add Lysis Buffer followed by Neutralizing Buffer, Centrifuge to pelletet protein precipitate, Transfer supernatant to                               GET Spin Column and                               centrifuge, Wash and elute                               plasmid DNA](https://static.igem.org/mediawiki/2021/3/35/T--Saint_Joseph--DNA-isolation.png)
* **Glycerol stock purchases**
  Prepared with 50% glycerin distilled water. The overnight grown cell culture is mixed 1:1 with
  50% glycerol. Store at -80 °C.

Next, we needed to insert the desired piece of DNA into our plasmids by enzyme cutting.

* **Recovery from Gel (with GF-1 DNA Recovery kit)**
  Digested DNA fragments separated and isolated from agarose gel with GF-1 DNA Recovery Kit
  according to the manufacturer’s instructions
  After cutting, we linked the additional DNA to our vectors by ligation.

![Ligation](https://static.igem.org/mediawiki/2021/d/d3/T--Saint_Joseph--DNA-Ligase.png)

![](https://static.igem.org/mediawiki/2021/2/2c/T--Saint_Joseph--Engineering-divider-3.png)

# Vector and Insert Preparation and Ligation

Depending on whether the insert DNA sequence is obtained as a DNA fragment or in the Cloning vector,
it is necessary to transfer this sequence to an Expression vector for the protein expression. With
this aim, the gene is cutted from the restriction enzyme sites (different from each other) placed at
the beginning and end of the sequence while designing the sequence, using appropriate enzymes. The
expression vector to which the sequence is to be transferred is also cut from the same enzyme cut
sites. Then, the insert gene ligated with the pET29b expression vector and adhered.

![Preparation of plasmids](https://static.igem.org/mediawiki/2021/f/fa/T--Saint_Joseph--Recombinant-Plasmid.png)

Once the desired result was achieved, it was time to screen the plasmids from the gel. We followed
the protocol in these parts.

Insert DNA plasmid backbones that isolated from the agarose gel were ligated with T4 DNA polymerase
and transformed into *E. coli* cells.

We ran our plasmids in agarose gel electrophoresis to see if the ligation was successful.

![Agarose Gel Electrophoresis](https://static.igem.org/mediawiki/parts/5/53/T--Saint_Joseph--Part-Agarose.png)

After ligation and transformation, transformant cells obtained at the LBA+Kan selective media. For
selecting the recombinant cells, transformant colonies are analyzed by colony PCR with T7 forward
and reverse primers, according to the tables below. After the colony PCR experiment, samples were
analyzed by agarose gel electrophoresis. And the obtained recombinant colonies inoculated into
LBB+Kan media overnight. After the incubation, plasmids isolated from these cultures. Isolated
plasmids were analyzed with cutting with restriction enzymes at the agarose gel.

Then, we again produced competent cells. Thus, we introduced our expression-ready vector into DH5a
and BL21 cultures by transformation so that it could grow.

* **Production of competent cell and transformation**
  In order to multiply the vector that is ready for expression, it must be administered in the E.
  coli DH5α strain. For the expression, the vector must be given to *E. coli* BL21(DE3) strain.
  Induction of protein expression and isolation
  For the targeted gene in the vector that has been administered to the expression strain to be
  expressed, protein expression must be triggered by the inducers that will join the environment.
  In these experiment protocols, usage of pET expression vectors that ensures high expression has
  been planned. In these vectors that the gene expression is executed under T7 promoter controle,
  IPTG or lactose can be administered in the medium as a trigger of gene expression.
  Underlying mechanism of induction can be explained:
  The lac repressor, constantly being produced by the lacI gene that is located in *E. coli*
  genome, inhibits the expression of T7 gene that executes the T7 Polymerase production by
  preventing the necessary polymerase in gene expression to stick to the gene. By the addition of
  IPTG in the medium, the IPTG attaches to the produced lac repressor and prevents the inhibition
  of T7 polymerase production; therefore, T7 polymerase is produced and gene expression under T7
  promoter controle can be executed.

  ![IPTG process](https://static.igem.org/mediawiki/2021/e/e3/T--Saint_Joseph--IPTG.png)

# Induction of protein expression

* **Production of competent cell and transformation**
  A colony is selected from the fresh petri and amplified under 30°C 2mL in LBB+Kan overnight.
  Overnight culture is diluted with LB+Kan by 1:50 ratio as it will be 5mL.
  It is produced in 37°C for 3 to 4 hours. Meanwhile, the medium that contains 1mL LB +
  antibiotic + 1mM IPGT is heated to 37°C. 1mL culture and 1mL LB + Kan + IPGT is mixed. It is
  incubated for 3-4 hours. Cells are separated by 1mL and collected by centrifuge. Pellet is kept
  until used at -20.

  As protein synthesis can
  be extremely fast in temperatures higher than 25-30°C, ratio of
  misfolded protein can increase. Optimisation is needed.

![Protein Induction](https://static.igem.org/mediawiki/2021/0/00/T--Saint_Joseph--Protein-Induction.png)

* **Protein Isolation**
  Protein isolation can be performed by the help of 6x Histidine Tag that is added to the 3’ end
  during the genetic design process. It is made by using Promega MagneHisTM Protein Purification
  System kit.

![Protein Isolation by magnets](https://static.igem.org/mediawiki/2021/a/a3/T--Saint_Joseph--Protein-Isolation.png)

![](https://static.igem.org/mediawiki/2021/c/ca/T--Saint_Joseph--Engineering-divider-4.png)

# Bradford Analysis

We used Bradford analysis which is used to determine the protein concentration.
In order to create Lyophilized BSA standard, 20mL deionised water is added and stirred until
dissolved/ If the standards will not be used within 60 days, they are aliquoted and stored in -20°C.

**Note:** Standards contain
the necessary buffer salts for the solubility of protein

For the quantification of pure enzyme samples isolated from *E. coli* BL21 bacteria, analysis was
performed using the Bradford method. During sample preparation for Bradford analysis, “Bio-Rad
Protein Assay Kit II” colorimetric protein measurement kit (Bio-Rad, 5000002) and BSA (Bovine Serum
Albumin) was used as a standard. A stock solution of 1 mg/mL BSA was obtained by adding 20 mL of
deionized water to lyophilized BSA. Five BSA standards were prepared at 0.1, 0.2, 0.3, 0.4, and 0.5
mg/mL concentrations by diluting the final volume to 100 microliters. To prepare the dye reagent
solution "Dye Reagent Concentrate" in the kit for spectrophotometric measurement, 400 microliters of
dye reagent was diluted with 1.6 mL of distilled H2O. 900 microliters of deionized distilled H2O was
added to each of the purified protein solutions containing the purified protein and having a final
volume of 100 microliters and these samples were used in the analysis. 10 microliters of each of the
BSA standard solutions and sample solutions were pipetted and loaded into the wells on the 96-well
microplate. Then, 200 microliters of diluted dye reagents were added to each well and pipetted until
all solutions became homogeneous. Each sample was loaded into the wells in duplicate. The microplate
was incubated for 10 minutes at room temperature. The microplate was placed in the "Thermo
Scientific Multiscan GO Microplate Spectrophotometer" device and absorbance was measured at 595 nm.
The BSA standard graph was created with the data obtained after the measurement. The protein
concentration of the samples was calculated by taking the BSA standard

# Enzyme activity control

pNP-cellobiose or carboxymethylcellulose can be used as substrates.

Enzymatic activity is predicted with the DNS method. The DNS method can be actualised with tracing
the reduction of sugars like carboxymethylcellulose (CMC). Reduction of glucose or other
oligosaccharides turns the DNS (yellow) to 3-amino-5-nitrosalicylic acid (orange-red). Change in
colour can be measured as absorbance with a spectrophotometer; thus, it can be used to determine the
activity of endoglucanase that produces the reducing sugars.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Saint Joseph/Results

![](https://static.igem.org/mediawiki/2021/4/4c/T--Saint_Joseph--logo-200.png)

[Project](/Team%3ASaint_Joseph)

[Description](/Team%3ASaint_Joseph/Description)
[Proposed Implementation](/Team%3ASaint_Joseph/Implementation)
[Contribution](/Team%3ASaint_Joseph/Contribution)
[Wiki Design](/Team%3ASaint_Joseph/Wiki)

[Human Practices](/Team%3ASaint_Joseph/Human_Practices)

[Education & Communication](/Team%3ASaint_Joseph/Communication)
[Inclusion](/Team%3ASaint_Joseph/Inclusivity)
[Education](/Team%3ASaint_Joseph/Education)
[SDG Impact](/Team%3ASaint_Joseph/Sustainable)
[Source Of Inspiration](/Team%3ASaint_Joseph/Source_Of_Inspiration)

[Engineering](/Team%3ASaint_Joseph/Engineering)

[Experiments](/Team%3ASaint_Joseph/Experiments)
[Results](/Team%3ASaint_Joseph/Results)
[Notebook](/Team%3ASaint_Joseph/Notebook)

[Team](/Team%3ASaint_Joseph/Team)

[Members](/Team%3ASaint_Joseph/Team)
[Attributions](/Team%3ASaint_Joseph/Attributions)
[Collaborations](/Team%3ASaint_Joseph/Collaborations)

[Parts](/Team%3ASaint_Joseph/Parts)

![]( data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAMIAAAC9CAYAAADhlaIlAAAACXBIWXMAAAsTAAALEwEAmpwYAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAABDbSURBVHgB7Z1/jBTlGcefGU5qU8SzsQbLYccUE6EaD9MImii7aVD7I+UuUJuQNOxpE/4xPdBA06bt3YkNCf6A0/7TpPWWNLGNhdzZNEohdvfaPwTTeGca0bQ0rOWI1Bp7gq2K7E7f590d2Dv2x+zuzLzvO/P9JOPt3i7e7M77eZ/neeeddywCwZMb7qaPPtVNl13WS5bVTVRyyLa/IF4Rj/k5eVstZiubwC3IxyV6i1zx2HVnqeu/05QeniUQKBaBznhxl0MLF6bIplvEM0d8pb2iATsULkIEa1r8nWkpSbGYp7t3TBNoG4jQKuWG31dp+H1Uv2dXgJWnUnFSyJGnddvzBHwDEZrBac75z/RSl71efF19EfT2AcJiuPvIPpen9A8KBOoCEWpxsfFvJu16/XaBFI2ACNUcfixV7vkpQ7Fo/PWoSPGVh7MEJBCBe//SFRmyLSGAm6JkURBbnujcSNKjRHJFYAHoykExtLmVYt37+yZLnxRHkzr6lDwRIEATRNp0vjiStFGn5IgAAVokWULEXwQI0CnZJNQQ8RbhpSdEEUx7CAIEQTbOQsRThNzuXqKuPQkcBQqbgjhrPRLHYdd4iVBOg4YqaRAIj4KIDuk4RQeb4gKfDKMrpiBBJDhEC09Qbs8QxQTzIwKigGoKcYgOZouQ2+WInilHsocCSinRNlE77CVDWUCmkntSDIku+I14tIQMpbvrU/T9626jiZvW0+V2F03OzpCxWHQvZe5xKJN+jfa9ZNyFQ+ZFhHIqJEaEShkymFR3D43deC85ly++8LvCR2dopPAyZU+/TgZTMDFVMkuEGKRC3PBZABahHnkRGQbePCjFMBbDUiVzRChPkR4nQ0+OcRo05NxOW3tu9f1vODJwhDBXCHuY0ttGyADMEEHWA66xhdigaPzDQgKWoVVYgtGZV2mv2AxlQggxIITQum7QXwQ5Vl0aJgPh9GfIuaNhGuQXw+uHgu51g94i5PaMmVgUcx2wZ3mK+q5eTkFjcLpU0FkGPUUwdGSIUx9Og7gOaCcNaoVhIcM+IYVhQhSIiv2U1u/iH/1EkBIsypXXBzKHzJIvyWK4ejg0bAxNl0StUEzrJoNeIhgoQZB1QLuwEOnp50yKDtrJoI8IhknQznBo2BhWP2glgx4iGCaBJ0DYdUA7zJ7/WA61shAGoI0MmojwZM6Ei2hqTYvQFYPqBy1kUC+CAUOkfqZF6Ioh0zUKqodW1Yqg+ckybziUzwqbjgH1Q0GlDOpE0FyCTqZF6Ir+6RIvdW+lVUzHUCNC7snNoibIkoboMBwaNizEtuN5mnj3OGnIBKUf7qeIiV6E8lTqKdJsFmmY0yJ0Rd90KfpZq9GKoOH1BFFOi9AVnq7BM1x56FUbbCtDax/aRxERsQiPT+l0roB7f44CJgyHho2G9UOkw6rRiaBRcZyEOqBdNJuuURChYVUUxXM0IpSXXhwjxeg4LUJXODJwQa0+XbLylH4oTSET/gJfXBfYpMVCUL2LroEEPuHZtL2LPkfqcVOiIw19zaoIVrrDukOgQ3ghZ7mebZh/IkzKSwI6BEDHLBgX7Sm0IffwRJBDpWZeawy0xBHtKbQUOxwR5LRqmRIBECRbKceLPQdPSBHhSqREICTssTBSpMBFGD7xsoOVqUGIOGGkSIGLMOSsyY3deA8BEBaifW113WAv5ApUBLFzMiXiMWjIAMKA2xW3L34o2ltgKVJgIoidcsSPYe85ZABBUyUB4xAXzwERZES4JG+DDCAo5kngMVjpgDsmEBHEzmTEj0yt1yAD6JQ6EjCcGgUyhy2oiNCwiucPMX7TNxM73x+0B7eXBhJ4pIIonDsWoRINnGbv47n/ud77IAPwBbcTbi9NJPDoeDi1IxEq+ZnvneDZjJABNMOToIXZr6lKh9w2nUaEzdTiGWTIABrRhgQeQ50Mp7Ytwvzh0laADKAWHUjAONTBcGonEaGjvAwygGo6lMBjsN2o0JYIlWiQoQ6BDIAJSAL5v6I2o0K7ESGwSU+QIdkEKIFHW1GhZRGCigbV8Jcw9eXvYFmVhMHHO2AJmLaiQjsRYZBCwPtSIEMyCEkCj5bbaEsiVEJOH4UEZEgGERzn7lbPK7QaEVgCh0IEMsSbCI/v5lbe3KoIkaxPBBniScTHtaU5SL5F8DunKCi8u9SA+KBgnVnfHXcrEaGlUBMEr33wDoH48Fb066mm/A6l+hKhMmSaoojR9EYWoE0m3v0HKcDXUKrfiKBk7dLpD/5NID5Mq4nwvk6w+RUhRRHDd4PU6sYVoGP4ePJxjRiWoOm6qU1FiLpI9kB9EE8mZ0+SAppmNH4iQuRFMoP6IJ4oiAhM06K5oQiqimRG0RcGQobrBEUpb8OiuVlESJECIEF8YQkUDYKsbfRiMxFCmWDXDEV5JIgIRce34ZnmuiJU0iIld8DMQ4RYozDip+q90CgihDbLtBGKhthAhHBHp6hOqDvw00iE9aQAnERLBoqOs1MvPaopgsrRoucxbJoIFB7nVK1f2q28OQqmcSItESiM/DVHj+qJoOQkGuqD5KCwTqh5cu0SESpvSpECMGyaLBSODmbm/6JWRFAyZMogGiSLSY2GUWuJoCQtYsLuIQofvS+2yC8OMRJOW8JOXRRGhEvqBG0iAjfQsAso/hvp6edo3+nXCdQnK76f64/8IvTjwf9/RXVC9/xh1DkiqDybHNW0a5Yh8+Yf5IFGKjYX/j64oxgQ309UDVSXYdT5EUFZfRD1tGsvOgy8eTDx6RJ/fv4e+PuIunPQZRh1vghKziYzqr6Q7OljMjqMFF6mpMG9Pn/uVX/5lfweVKDwupM5nb4WESGK+qAZw6JBsBBJqR+452cB+HOrvCSWj70OdcIFESrnDxI929SrH7iBxDVd4g6HUyDedPmMz2sQFexav4wa3U6kcWPh6BCn+oF73W3H81Jy3QYJFO7PLd6DrqpfpkgRuo7ecN7M+8Z3dhxybidTGZ15VXkK1AiFGUHKe2B5D0RqlCMFMnCPy72v7vBShcNChs3+bneqBSzxtuM5I6a2n1jzXVVr3V5lWdas8tTIlKvRvPrBhHTJGxrmzZTrO1SfT5AiVE6ktX1rzk4w7foDb7iVe1rdhKgeDjXtZKFCYR3+j139RAWmXpG2d2ZKq+ka3rQInWuBRig8nyALZk8EJWkRS2DyqEz1dA1VQquYFhEGvO+K2kKK/yOLZZEajVNEF+vzh+V0iHsAhZOuQiGzZKUYXbojkqKvPByaU3ZGOAz4Dpu9i66R3+Pa7mVRFs9XeSJMUUhRwVvQyWv8SZjXw6NLYQ238vfJw6F7xRb3RZJZhFR3D62/ern4uSzMWxCv8kT4DwVYLMe51/dLGMOtnAYleZIgy9B39RdltAj4bpwDVmXE6AR1QPla45PyiiMu2rCc+0X4gI3ftL6jMO/NDsW08YsEHC1GrMrEoxy1CB8UnhqRlxsOUDPaqR+84VBOg0BjOowWWaty/4OxZu/kg8KpDvf6/BO9fuuwBFt7bqVBsTVD92kROuNFC5bDZ9E9zSIMU50bKaDXD4dG9YNJ0yJMgYVgMdZWftagwCJkqXLBPnr9aKlOl1AHRAPXEixDn6gtqqOF9bt3jmf/+P7JzdzroxdSA+e2uGecGrie4Ihh0UuP7yXbUnIfBAA0gWefugUCING4BZtsmiYAEo01LSJCF4swSwAkl0mb0ttm2QgCILHY+co07OIIAZBMsiIYFMoipLfnRVTIEwCJw5ZBoOqaZUQFkDhkNOAHF0XgqFByRwmAZFDwogEzd8lHe8EwCmeQDOx+LxrIZ3NeK48g9ZO0BYC4UhwQbX1Oh3/pjUKkJXaaIAOIH7NlCXZk579Q+66aZRlWoWYAMaIgO/gaEjBW03+eeyJD5esVHALAPEQUKIkOvWtvOfWvTXMRPKQQ7qD4J8pWzQagBXwJ4OFfBI/cHkf8AV4Daa0Qw4EYQBNEY3enqUSvke1OlE8S+6d1EWqR2+VQCPzNuSF3mYWULC4c+t/Z/i3/Oh3C8Pzls356/UYEI0JIuKcmTohddAjEg1IpbS3rz5OG2AQAgAgAMBABAIIIAEggAgAEEQCQQAQAaO59lkEDzpz9kA68+Aod+/spmjn9nvzdmlXLacNXb6Oeaz9LKuF9G/vtpNy3Mx98SIsXfZruvvNmWi32T/W+mQJOqPlg7LlJGn3moGxktWAZfjzYLxtg1Ox8alzuXz0G779Xblqg8Qk1RIQmsAC8NYIjxdGp4/T77PZIZfhG5jE6dvxUw/d4+66NDJoCERpw4IVX5kjQ09NDmUyGVq5cWX79wAE6dOgQnT17VqZL23/6LP181wMUBbxf1RKsWbOGNmzYQEuXLqUzZ87Q4cOH5f5572VBB+5bS6A2SI0acNfGRy7UAxs3bqTdu3df8p6ZmRnatGmT/Mk8+/SDsnYIk5m336O7vvXIheeDg4Nym8/+/ftpx44d8jGL8Kf9P1GSvl0Ac43Mo7oo5khQS4Jarx3+818pbKr/BkeBWhIwLK/3Gtc3/JlAbSBCHU5VJGAGBgYavpfTktWrV8vHUTS2I1MX71LfbN+qXz86pezu9toDEepQPUK0YsWKpu/nyMCcevs9CpvqffPqlXosXrz4wr6B+kAEAAgiACCBCAAQRABAAhEAIIgAgAQiAEAQAQAJRPABT2ID8QYi+OCNN94I5D1hcOTIkYav82RAb0IgqA9E8MHY2FjDqMBTsY8dO0Yq8KZat/s6KAMRfMASbNmypWbPygI8+uijpApu6KOjtW9jwQLXew3MBRfm+OTo0aPyuoN169bJ2aYsB6clOvS43Nh5P3imKU+wY2E5SvE+A39AhBbgBsa9LG+6wfu2c+dOAu2B1AgAgggASCACAAQRAJBABAAIIgAggQgBE8W6QUrXJoopEKEOS5e0t3ju0ggW3W13Yd92P1MSgAh1WHnDUrqijZ533Z03U9i0+zfW3BruCnwmAxHqwOnH/S2uFdojetyNX7uNwoaXlFwhRG0FuXw9IkJdIEIDeNHcVhrPj77XT1Hx2A83+Y5Y/BkGH8Bq2I2ACA3gqPDszx5sKgO/jxvm3XeFnxZ5cOr266eb7xu/7uczJB2shu2T/S+8Qk89c/DCwsAMC8Apx8C31ypraLw/o788KBcGrl4K0lsGnjdtRpk0Xg0bIrSIt6o0N3zdbsvk3TpKx32T4I458YF717Dvf9AuK1ssoMFF9K4RLLtAID4sWHiCNEVvEUrFtwjEBuvzX9f2eOo+ajRNIC5ofSz1FsGFCPHBgghtc16KMEvAfNziJGmM1iJY1/fPkqV3TwJ88gnlSWP0P7NcLI4QMJzS86JTK5DGaC9C5QQM0iOTKVKWNMeMuUalEpZrM5eCdV3/BGmOGSKcp72EqGAmbsmI1NYIEWTR7Ja2ETAMd8Lq6c+SARgzDVt+oZaVJ2AKs3TONabzMut6hI+LfOVLgYD+iAiu+0hRNUaJUEmRWAbUCzpTKo2YkhJ5aH09Qj3cf4730QJ7nIB+uKV9QoIMGYaRl2rK4bgiIoN2iGFuEyVgjIwIHu7b4w6V7Jx46BBQC6dDy/qHyVCMvnjfulYUY3YpzeGYgCoKlUswh8lgjI4I1bgz4xmy7CFCdIgOPuN/noblIIbhxEYEDwgRBVaWzhVHTBoebUbsRPAojywt2CwepcTTbgKd4bp5sU3ydJc4RID5xFaEatyT4ynxSXvJWnALuRwpSt3iOeSohytG4yx7mkrF98XjPBUpH8fGX83/AYZVqjkejyobAAAAAElFTkSuQmCC)

# Loading

# Results

![](https://static.igem.org/mediawiki/2021/2/2a/T--Saint_Joseph--Engineering-Jambatron-bg.png)
![](https://static.igem.org/mediawiki/2021/2/2a/T--Saint_Joseph--Engineering-Jambatron-btm.png)

# Bioinformatic Design Results

Genes of the enzymes have been cloned to the pET29b expression vector between NdeI and XhoI
restriction sites.

![](https://static.igem.org/mediawiki/2021/4/49/T--Saint_Joseph--plasmids.png)

![](https://static.igem.org/mediawiki/2021/5/56/T--Saint_Joseph--Engineering-divider-2.png)

# Creation of Recombinant DNA

The gene was cut from the enzyme restriction sites which are different from each other
using appropriate enzymes. 1μL restriction enzyme was used. The expression vector, which has been
used as backbone, also digested from the same restriction sites. Then the gene sequence is ligated
with the backbone. The enzyme was added last on ice and an enzyme buffer that is suitable for both,
was used.

![Agarose Gel Electrophoresis](https://static.igem.org/mediawiki/parts/5/53/T--Saint_Joseph--Part-Agarose.png)

First, we measured the mass of the gel. Then added Buffer GB at a 1:1 ratio, assuming a volume of
100μl per 0.1 gram. Next, we centrifuged the tube to ensure that the gel has descended into the
Buffer. We incubated the gel at 50 °C until it melted and, for a better result, we inverted the tube
from time to time. We passed the liquid to the Column in the collection tube that comes in the kit
(We used GF-1 DNA Recovery kit). We centrifuged it at 10000 x g for 1 min. Then, we discarded the
liquid in the collection tube. We added 650μl Wash Buffer to the colon, centrifuged at 10.000 rpm
for 1 min. We discarded the liquid in the collection tube one more time.

We centrifuged the empty column one more time to remove any ethanol residue.
Next, we did ligation. The mix contained: T4 DNA Ligase Buffer, Vector DNA, Insert DNA,T4 DNA Ligase
and water. We mixed the reaction by shaking it up and down. For the sticky ends, we made an
incubation process for 10 minutes at room temperature. It should have been deactivated by preserving
it at 65 °C. Then we cooled it on ice. We ran the end product on agarose gel on convenient voltage
and duration

![](https://static.igem.org/mediawiki/2021/2/2c/T--Saint_Joseph--Engineering-divider-3.png)

# Protein Expression

Induction was done with IPTG. This was done at a density of 30 μmoles and 50 μmol. The last
concentration was 0,5 μmol. Right after IPTG, protein isolation was done. At the end of the
isolation, we did a Bradford Analysis which determined the concentration of the enzyme

CelAB, EGII and EGII (C99V) protein concentrations were observed as 0.127, 0.1 and 0.102,
respectively.

![](https://static.igem.org/mediawiki/2021/6/69/T--Saint_Joseph--Bradford-Assay.jpg)

|  | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| A | Standart | Standart | Standart | Standart | Standart | Standart |
| B | CelAB | CelAB | CelAB | EGII | EGII | EGII |
| C | EGII C99V | EGII C99V | EGII C99V | - | - | - |

| Enzyme | Absorbance | Concentration (mg/mL) |
| --- | --- | --- |
| CelAB | 0.3726625 | 0.127356103179867 |
| EGII | 0.2968375 | 0.3006875 |
| EGII (C99V) | 0.100889210792698 | 0.102233062236029 |

![](https://static.igem.org/mediawiki/2021/c/ca/T--Saint_Joseph--Engineering-divider-4.png)

# Enzyme Activity Determination

To test the enzymes we conducted a CMCase activity analysis test. Briefly, we prepared standards
that we know their glucose concentrations and measured their absorbance at 540 nm after DNS
treatment.
Depending on the concentration-absorbance, we made graphs. We then measured the absorbance of the
samples we treated with the enzymes. We calculated the amount of product we obtained with the
enzymes by substituting the absorbance values ​​we obtained in the equation we obtained from the
standard graph. Then, by drawing the product formation graph against the dilution amount, we
calculated from the graph equation how much product we could produce when we use the enzyme without
dilution. We wrote the amount of product we obtained in milligrams in μmol. We have calculated the
Enzyme Activity by taking into account the reaction time.

Different from the CMCase Enzyme Activity protocol we diluted 2 enzymes as 100-times and
1000-times.

* For CelAB, we got 189,3 mg product yield. This quantity is equal to 1051 µmol
  product formation. The unit value is 17,5 U/min.
* For EGII, we got 268,5 mg product yield. This quantity is equal to 1491 µmol product formation.
  The unit value is 24,85 U/min.
* For EGII(C99V), as shown (Figure 4) we got 208,24 mg product yield. This quantity is equal to
  1156 µmol product formation. The unit value is 19,26 U/min.

All of the enzymes got pretty good enzymes activities. Wild type EGII got the highest U/min activity
and CelAB got the lowest activity with 17,5 U/min.

Even though we proved enzyme’s activities, the activity results can be enhanced with optimization of
protein induction and substrate-enzyme treatment conditions.

# Demonstration of CelAB, EGII and EGII C99V Enzyme Effects on Filter Paper

CelAB, EGII and mutant EGII C99V genes originating from the specified organisms were inserted into
the pET29b(+) plasmid vector, and enzyme production was performed by inducing lactose in
Escherichia coli BL21 host organism. CelAB is a cellulase type enzyme that breaks down the cellulose
molecule, which is a polysaccharide, into monosaccharides such as β-glucose. EGII is a glucanase
type
enzyme and is also responsible for the depolymerization of cellulose, glucose is released as the end
product. Glucose gives its highest absorbance in the wavelength range of 260-270 nm.
Filter papers in the laboratory are used to see the effectiveness of enzymes that break down
cellulose,
since they are in cellulose structure. In the experiment, filter papers of equal size and
approximately
equal weight were placed in enzyme lysates with equal concentrations, the control group included the
enzyme-free lysate solution (Table.1). The tubes were incubated for 72 hours at 50°C with shaking.
The
appearance of the tubes after 20 hours and 72 hours (after removing the filter papers) is shown in
Figure.1 and Figure.2, respectively.

**Table.1:** Enzymes used in the experiment, their concentrations and
filter papers

| Enzymes Samples | Enzyme concentrations | Filter paper size | Filter paper weight |
| --- | --- | --- | --- |
| Control | - | 1x6cm | 37.9mg |
| CelAB | 3 mg/ml | 1×6 cm | 35 mg |
| EGII | 3 mg/ml | 1×6 cm | 39.6 mg |
| EGII (C99V) | 3 mg/ml | 1×6 cm | 37.3 mg |

**Figure.1:** Image of test tubes after 20
hours
![](https://static.igem.org/mediawiki/2021/4/4c/T--Saint_Joseph--Parts-Results.png)

**Figure.2:** Image of test tubes after 72
hours
![](https://static.igem.org/mediawiki/2021/2/22/T--Saint_Joseph--Parts-Results2.png)

In the qualitative determination, the depolymerization effect of the enzymes on the filter paper is
observed by looking at the images of the tubes and the pieces of paper accumulated at the
bottom.

In the quantitative determination, the pre- and post-experiment masses of the papers used as the
substrate, the shredded paper masses accumulated in the tubes, and the glucose end product
absorbance in the liquid in the tubes were measured using a
spectrophotometer.

Spectrophotometer measurements were made triplicate at 260 and 270 nm
wavelengths, the raw
absorbance data are given in Table.2. The absorbance value of the released glucose depending on the
samples is shown in Graph.1.

| Samples | λ 1 | Abs. 1 | λ 2 | Abs. 2 |
| --- | --- | --- | --- | --- |
| Blank 1 | 0,000 | 270 | 0,000 | 260 |
| Control | 0,036 | 270 | 0,055 | 260 |
| Control 1 | 0,057 | 270 | 0,077 | 260 |
| Control 2 | 0,050 | 270 | 0,067 | 260 |
| CelAB | 18,48 | 270 | 22,50 | 260 |
| CelAB 1 | 20,31 | 270 | 22,50 | 260 |
| CelAB 2 | 18,61 | 270 | 22,50 | 260 |
| EGII | 14,57 | 270 | 16,97 | 260 |
| EGII 1 | 14,26 | 270 | 16,60 | 260 |
| EGII 2 | 14,36 | 270 | 16,77 | 260 |
| EGII C99V | 16,47 | 270 | 19,10 | 260 |
| EGII C99V 1 | 17,18 | 270 | 19,91 | 260 |
| EGII C99V 2 | 16,99 | 270 | 19,66 | 260 |

As seen in the graph, the highest glucose absorbance and thus the highest glucose end product
formation was observed in the treatment with CelAB enzyme. It can be said that CelAB has the highest
cellulose degradation capacity among the three enzymes. EGII was found to have lower efficacy than
CelAB. It was observed that the ability of the EGII C99V enzyme, which was obtained as a result of
the mutation in the EGII enzyme, in cleaving glucose monomer from cellulose structures improved
compared to wild type EGII and approached CelAB, as expected.

The before and after mass of filter papers used in the experiment were weighed. Weighing results are
given in Table.3. Despite drying in an incubator at 50°C and room temperature for one week and in
microwave for 20 seconds, the post-experiment mass of the papers was higher than before the
experiment. It was observed that the solution and enzyme used in the experiment could not be
completely removed from the papers by these drying methods.

**Table.3:** Masses of filter papers before and after the
experiment

| Samples | Pre-experimental mass of paper (mg) | Post-experimental mass of paper (mg) |
| --- | --- | --- |
| Control | 37,9 | 42,3 |
| CelAB | 35 | 44,4 | 0,055 | 260 |
| EGII | 39,6 | 45,2 |
| EGII C99V | 37,3 | 45,3 |

Despite the fact that the mass of the papers increased, the sedimentation masses formed by the
shredded papers in the tube were compared, since fragmentation was clearly observed in the tubes.
After the papers were taken from the test tubes, the tubes were centrifuged and the liquid part was
discarded, so the mass of the precipitates was determined by comparing it with the mass of the empty
tube. The results are given in Table.4 and Graph.2.

**Table.4:** Masses of tubes and sediments accumulating at the bottom
before and after the experiment

| Samples | Empty tube mass (g) | Tube mass after test (g) | Mass of precipitate (g) |
| --- | --- | --- | --- |
| Control | 6,9306 | 6,9328 | 0,0022 |
| CelAB | 6,7575 | 6,8782 | 0,1207 |
| EGII | 6,9053 | 6,9686 | 0,0633 |
| EGII C99V | 37,3 | 45,3 | 0,0403 |

**Graph.2:** Masses of shredded paper precipitates according to
samples

The precipitated paper masses plot gives proportionally consistent results with the
spectrophotometer
measurements for CelAB and EGII samples. Again, the highest degradation, that is, the highest enzyme
activity, was observed in CelAB. Unlike the spectrophotometer results, the amount of paper degraded
by the EGII C99V enzyme was the lowest. The reason for this is the possibility that, due to the
mutation
in the enzyme structure, it exhibited the behavior of cleaving the final product, glucose monomers,
rather than completely physically breaking down the substrate compared to other enzymes. Another
reason may be an experimental error that occurred while getting rid of the liquid portion after
settling.