iGEM 2021 — Team UBrawijaya

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# Team:UBrawijaya/Notebook

Team:UBrawijaya - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/4/4c/T--UBrawijaya--Logo.png)](https://2021.igem.org/Team%3AUBrawijaya)

* Project
  + [Description](https://2021.igem.org/Team%3AUBrawijaya/Description)
  + [Implementation](https://2021.igem.org/Team%3AUBrawijaya/Implementation)
  + [Safety](https://2021.igem.org/Team%3AUBrawijaya/Safety)
  + [Sustainability](https://2021.igem.org/Team%3AUBrawijaya/Sustainable)
  + [Entrepreneurship](https://2021.igem.org/Team%3AUBrawijaya/Entrepreneurship)
* Labwork
  + [Engineering](https://2021.igem.org/Team%3AUBrawijaya/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AUBrawijaya/Notebook)
  + [Proof of Concept](https://2021.igem.org/Team%3AUBrawijaya/Proof_Of_Concept)
* Dry lab
  + [Parts](https://2021.igem.org/Team%3AUBrawijaya/Parts)
  + [Contribution](https://2021.igem.org/Team%3AUBrawijaya/Contribution)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AUBrawijaya/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AUBrawijaya/Education)
  + [Science Communication](https://2021.igem.org/Team%3AUBrawijaya/Communication)
* Team
  + [Members](https://2021.igem.org/Team%3AUBrawijaya/Team)
  + [Attributions](https://2021.igem.org/Team%3AUBrawijaya/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AUBrawijaya/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AUBrawijaya/Partnership)
* Awards

## Notebook

## August 25, 2021 - September 10, 2021

**CULTIVATION & PRESERVATION MICROBE**- Cultivating DH5 alpha/BL21/BL21 (DE3) from the broth medium to the slant agar for
  storage

Protocol:

1. Sterilize the ose and put it into a briefly vortexed cultivation broth
2. Streak the ose into agar
3. Incubate 12-18 hours 370 C in an incubator
4. Store on the refrigerator at 40 C

- Cultivating DH5 alpha/BL21/BL21 (DE3) slant medium into broth medium

Protocol:

1. Sterile the ose third time with alcohol 70% and burnt the ose on fire (bunsen) until the ring is changed to the red
2. Collect one colony on slant medium and transfer into 30 mL LB broth as a starter for the next step of competent cell protoco
3. Incubated 12-18 hours 370C in incubator or shaker waterbath using 120 rpm 370C
4. Incubated 12-18 hours 370C in incubator or shaker waterbath using 120 rpm 370C

* Preservation recombinant DH5 alpha/BL21/BL21 (DE3)

Protocol:

1. CTransfer 400 µL culture and add 200 µL glycerol 60% into the eppendorf tube sterile (final concentration 20% preservation)
2. Gently pipette
3. Store on deep freezer (-80oC)

## September 11, 2021 - September 30, 2021

**PCR**- Resuspended Primer IDT

Protocol: base on IDT protocol

1. Prior to opening, centrifuge the tube at a minimum of 3000xg to ensure that the material is at the bottom of the tube
2. Add TE buffer/dH2O (distilled water)/NFW (nucleus free water) which amounts to …. µL that calculated from nmole of the primer (\*each primer have different nmole) you able to use calculator IDT tool in https://sg.idtdna.com/calc/analyzer and choose resuspension calculator tool
3. If you want to dilution the shock you able to calculated on the same link and choose dilution calculator
4. Mix the TE buffer/dH2O/NFW and primer stock on a sterile microtube
5. Serial dilution (optional)
6. Storage -200 C

#### Label A

Forward plasmid backbone pSB1C3 & pSB1K3
27.2 nmoles + 272 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

#### Label B

Reverse plasmid backbone pSB1C3 & pSB1K3
26.9 nmoles + 269 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

#### Label C

Forward (1) backbone overhang pSB1C3
26.5 nmoles + 265 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

#### Label D

Reverse (1) backbone overhang pSB1C3
19.4 nmoles + 194 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

#### Label E

Forward (2) insert pSB1C3
29 nmoles + 290 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

**Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base**

#### Label F

Reverse (2) insert pSB1C3
24.2 nmoles + 242 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW
**Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base.**

#### Label G

Forward (1) backbone overhang pSB1K3
22 nmoles + 220 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

#### Label H

Reverse (1) backbone overhang pSB1K3
22.8 nmoles + 228 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

#### Label I

Forward (2) insert pSB1K3
27.8 nmoles + 278 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

**Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base.**

#### Label J

Reverse (2) insert pSB1K3
26.5 nmoles + 265 µL NFW → stock concentration 100 µM
Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

**Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base.**

- Resuspende gblock insert pSB1C3

Protocol: base on IDT protocol

1. Prior to opening, centrifuge the tube at a minimum of* 3000xg to ensure that the material is at the bottom of the tube
   * Add TE buffer/dH2O/NFW to reach a final concentration 10ng/µL
   * Vortex briefly
   * Incubate at 50oC for 20 minutes
   * Briefly vortex and centrifuge
   * Serial dilution (optional)
   * Storage -20oC

* PCR reaction

Protocol: base on NEB protocoll

1. Mix 25µL volume reaction, add
   10 µL Forward (FWD) (label E) 1.25 µL
   10 µL Reverse (REV) (label F) 1.25 µL
   Template DNA 1 µL
   NEB Q5 master mix 12.5 µL
   NFW 9 µ+ Spin down for a second
   + Put PCR eppendorf into PCR mechanine
   + Setting condition PCR 30 cycles
     98oC 30s
     98oC 10s
     ? 30s → annealing temperature depends on primer
     72oC 30s
     72oC 2 mins
     4oC 5 mins

**note: if running gradient PCR annealing temperature using multi-temperature (56oC-72oC ) to get optimal annealing temperature**

## September 11, 2021 - September 30, 2021

**CLEANUP PCR**- DNA PCR cleanup using kit NEB [PCR & DNA Cleanup Kit (5µg)]

Protocol: Base on NEB protocol

1. Dilute sample with DNA cleanup binding buffer 5 : 1 sample [dsDNA < 2 kb] for amplicon product then mix well by pipetting (do not vortex)
2. Insert column into the collection tube and load sample onto the column
3. Spin 13.000 rpm 1 minute, then discard flow-through
4. Re-insert column into the collection tube and add 200 µL DNA wash buffer
5. Spin 13.000 rpm 1 minute, then discard flow-through
6. Repeat steps 4-5
7. Repeat steps 4-5
8. Add 20 µL of DNA Elution buffer to the center of the matrix and wait 1 minute
9. Spin 13.000 rpm 1 minute, then remove the filter tube and DNA elute on the 1.5 mL microfuge tube
10. Measure concentration and purification DNA with nanodrop

## September 11, 2021 - September 30, 2021

**ELECTROPHORESIS**- Electrophoresis DNA

Protocol:

1. Made 1.5 % agarose gel and add 1x TBE buffer solves then heat it around 4-5 minutes next pour into the electrophoresis plate print and wait until the gel hardens
2. Prepare and mix components for marker well:
   Ladder 1 Kb 4 µL
   Gel red 2 µL
3. Prepare and mix components for the sample well:
   Loading dye 2 µL
   Gel Red (DNA staining) 2 µL
   DNA (>100 ng/µL) 1-5 µL → PCR product
4. Transfer the gel into the electrophoresis equipment and pour the 1x TBE buffer until the gel submerged
5. Loading the marker and the DNA sample into the well
6. Running by 50 volts for 60 minutes
7. Visualized the gel with gel dock

## September 11, 2021 - September 30, 2021

**PLASMID ISOLATION**- Plasmid isolation using NEB kit [Monarch plasmid miniprep kit]

Protocol: base on NEB protocol

1. 1 mL - 5 mL culture do centrifugation for 30 seconds 13.000 rpm and get the pellet for isolation
2. Resuspended pellet with add 200 µL plasmid resuspension buffer (B1) then pipet up and down to ensure cells are completely resuspended
3. Add 100 µL plasmid lysis buffer (B2) and gently invert the tube around 5-6 times, then incubate 1 minute at room temperature
4. Add 400 µL plasmid neutralization buffer (B3) and gently invert the tube until neutralized, then incubate 2 minutes at room temperature
5. Centrifuge lysate for 2-5 minutes 13.000 rpm
6. Transfer supernatant to the spin column and centrifuge for 1 minute 13.000 rpm, discard flow-through
7. Re-insert column into the collection tube and add 200 µL DNA wash buffer 1 and centrifuge for 1 minute 13.000 rpm, then discard flow-through
8. Add 400 µL plasmid wash buffer 2 and centrifuge for 1 minute 13.000 rpm
9. Transfer column to a clean 1.5 mL microfuge tube
10. Add 30 µL of DNA Elution buffer to the center of the matrix and wait 1 minute
11. Spin 13.000 rpm 1 minute, then remove the filter tube and DNA elute on the 1.5 mL microfuge tube
12. Measure concentration and purification DNA with nanodrop

## October 4, 2021- October 21, 2021

**ASSEMBLY & TRANSFORMATION**- Gibson Assembly and Transformation [NEBuilder HiFi DNA Assembly Cloning Kit]

Protocol: base on NEB protocol

1. Set up following reaction on ice
   Insert DNA length = …...bp
   Vector DNA length = …...bp
   Vector DNA mass = …... ng/µL → 1 µL
   (required insert DNA mass …...ng [3:1])
2. Calculation of how much µL for fulfilling required insert DNA mass …..ng → …..µL
3. Add 2-3 fragment assembly with recommended DNA ratio (3:1) and mix all reactions with pipetting
   Total amount fragment X µL
   NEBuilder HiFi 10 µL
   NFW 10-X
   Total volume 20 µL
4. Incubated the sample in a thermocycler at 50oC for 15 minutes and then storage at -20oC for a moment or around 15-30 minutes
5. Thaw chemically competent cells on ice
6. Add 2 µL of the chilled assembled product to the competent cells, then mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex
7. Place the mixture on ice for 30 minutes. Do not mix
8. Heat shock at 42oC for 30 seconds. Do not mix
9. Transfer tubes to ice for 2 minutes
10. Add 950 µL of room-temperature SOC media for DH5 alpha and LB media for BL21/BL21 (DE3) into the tube
11. Incubate the tube at 370C for 60 minutes. Shake vigorously (250 rpm) or rotate
12. Warm selection plate to 370C
13. Spread 100 µL of the cells onto the selection plates. Use CHL plates for the positive control sample
14. ncubate overnight (12-18 hours) at 370C

- screening selection media

Protocol:

1. Pipet antibiotic chloramphenicol 33 µg/mL or kanamycin 30 µg/mL (1 mL of medium add 1 µL of antibiotic)
2. spread the mixture evenly across the plate
3. Incubate at 37°C overnight or approximately 12-18 hours

[![](https://static.igem.org/mediawiki/2021/4/4c/T--UBrawijaya--Logo.png)](https://2021.igem.org/Team%3AUBrawijaya)

### Follow us

### Contact us

* igemub2021@gmail.com
* Jl. Veteran, Malang, East Java 65145

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

# Team:UBrawijaya/Engineering

Team:UBrawijaya - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/4/4c/T--UBrawijaya--Logo.png)](https://2021.igem.org/Team%3AUBrawijaya)

* Project
  + [Description](https://2021.igem.org/Team%3AUBrawijaya/Description)
  + [Implementation](https://2021.igem.org/Team%3AUBrawijaya/Implementation)
  + [Safety](https://2021.igem.org/Team%3AUBrawijaya/Safety)
  + [Sustainability](https://2021.igem.org/Team%3AUBrawijaya/Sustainable)
  + [Entrepreneurship](https://2021.igem.org/Team%3AUBrawijaya/Entrepreneurship)
* Labwork
  + [Engineering](https://2021.igem.org/Team%3AUBrawijaya/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AUBrawijaya/Notebook)
  + [Proof of Concept](https://2021.igem.org/Team%3AUBrawijaya/Proof_Of_Concept)
* Dry lab
  + [Parts](https://2021.igem.org/Team%3AUBrawijaya/Parts)
  + [Contribution](https://2021.igem.org/Team%3AUBrawijaya/Contribution)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AUBrawijaya/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AUBrawijaya/Education)
  + [Science Communication](https://2021.igem.org/Team%3AUBrawijaya/Communication)
* Team
  + [Members](https://2021.igem.org/Team%3AUBrawijaya/Team)
  + [Attributions](https://2021.igem.org/Team%3AUBrawijaya/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AUBrawijaya/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AUBrawijaya/Partnership)
* Awards

## Engineering Success

Demonstrate our engineering success in a part of our project.

[Check Below](#blog)

The iterative engineering process is the core method used by any iGEM team to create their final
product. During our brainstorm phase, we have known that we wanted to make an overexpression system for
enzymes as enzyme yield problem is still an obstacle in national enzyme industries. We thought we would
be able to test the parts in the registry utilizing the outer membrane proteins, specifically the eCPX.
But the eCPX available on the registry lacks the linker that we need to attach our enzymes of interest,
in this case the blue chromoprotein amilCP. Therefore, we have to design our own circuit to fulfill our
goals for this project.

We readjusted our plans and priority plenty of times to ensure the maximum efficiency for our system and
to ensure that our system is working properly. We also had several revisions, going through the DBTL
iteration cycle plenty of times.

Aside from the technicality based on our own reflection that we need to improve our system, our
integrated human practices also play a huge role in our iteration cycle. The needs for a higher yield
expression system while still considering an easier and cost-effective system becomes a priority that we
need to do.

### Design 1:

On the first design stage, we designed two different kinds of constructs in two different standard iGEM
plasmids, the pSB1C3 and the pSB1K3. As the proof of concept, we use the blue chromoprotein gene
(BBa\_K592009) as our protein of interest. Because we want to make a fusion of the outer membrane protein
or eCPX with the PoI, we insert the TEV protease-specific linker site and the eCPX gene sequence after
the blue chromoprotein. We also added a strong universal promoter at first because in the beginning of
our project, only BL21 is available in our lab but we still kept looking for BL21(DE3) for higher
recombinant protein expression. We also added double stop codon at the end of our design.

The considerations when we design our circuits are:

1. Producing higher yield of recombinant protein expression, in this case the amilCP blue chromoprotein
2. The fusion between the eCPX and the amilCP is connected by a TEV protease-specific linker. This
   allows only TEV protease to be able to cleave the site and detach our amilCP, only when we want them
   to be cleaved.

![](https://static.igem.org/mediawiki/2021/7/7e/T--UBrawijaya--Labwork--Engineering--diagram1amilCP.jpeg)

Because we need TEV protease for our specific linker, we also needed to design another construct
containing the TEV protease gene itself. We are using a different plasmid than the first one. For the
TEV protease, the pSB1K3 plasmid which has the resistance for kanamycin antibiotic is used.

The sequence inside the pSB1C3 with chloramphenicol resistance is the eCPX-amilCP fusion. At first, we
were planning to integrate our system to several microorganisms, those are the E. coli BL21, E. coli
BL21(DE3), E. coli JW0729-3 with a hypervesiculating ability, and the C. violaceum (from iGEM
UGM\_Indonesia) to test our system into another chassis and also as a form of our partnership with the
UGM team. But due to the time limit, we were only able to test our systems on the E. coli BL21 and
BL21(DE3) yet we are still hoping to be able to express our systems on the strains in our initial plan
for the future prospect.

While waiting for the T7 promoter specific for BL21(DE3), we were using the universal expression of
promoter.

![](https://static.igem.org/mediawiki/2021/1/1a/T--UBrawijaya--Labwork--Engineering--TEV_universal.png)
![](https://static.igem.org/mediawiki/2021/d/d9/T--UBrawijaya--Labwork--Engineering--Sec_universal.jpeg)

### Design 2:

Synthesizing both genetic circuit with T7 promoters in order to try it in the E. coli BL21(DE3).

![](https://static.igem.org/mediawiki/2021/0/03/T--UBrawijaya--Labwork--Engineering--diagram2Sec.jpeg)
![](https://static.igem.org/mediawiki/2021/6/63/T--UBrawijaya--Labwork--Engineering--amilcp_T7.png)
![](https://static.igem.org/mediawiki/2021/d/dd/T--UBrawijaya--Labwork--Engineering--TEV_T7.jpeg)

### Design 3:

All of our gBlocks have the right length yet we still haven’t been able to properly assemble the insert
to the plasmid backbone. This is analyzed from the presence of colonies that is grown on the petri dish
yet do not release the colour blue as it is supposed to be due to the insertion of amilCP gene sequence.
We redesign our primers for the insert to be more specific.

### Design 4:

Addition of his-tag to facilitate easier protein purification.

On the testing stage, we induced the transformed colonies with lactose in the hope that it will produce
the colour blue. The test will be successful if the spectrophotometer shows the colour blue is present
on the media along with the cells. This confirms that the cells produce the blue color from the eCPX-EoI
fusion. While to confirm whether the EoI is still intact with the eCPX, we might need to do an SEM and
TEM analysis. But due to the time limit, we might not be able to conduct this.

![](https://static.igem.org/mediawiki/2021/b/b3/T--UBrawijaya--Labwork--Engineering--Projectdescription.png)

From the picture, there are separated fractions, the vesicles and the pellet. The vesicle fraction that
is clean can go through selective release. Thus, the yield would be much cleaner. The pellet can be
added with TEV protease for selective release of outer membrane protein still on the cell membrane
(on-pellet digestion). The on-pellet digestion is also a highlight of our project as it needs only a
minimal amount of media just sufficient for resuspending the cells. The live cells from this process can
be reused for another process.

The use of eCPX-PoI fusion would also allow for protein localization both in the cell membrane or in the
vesicles shed by the cells as their natural mechanism. By overexpressing the eCPX, we can make the
protein attached to the eCPX to be concentrated on the cell membrane and carried out onto the vesicles
when shedding.

There are two ways enzymes can be produced, intracellularly and extracellularly. Intracellular enzymes
would be more difficult to recover as it would need cell disruption and need to be separated from the
cellular impurities. Enzymes produced intracellularly would also be limited due to the host size and
space inside of the cells. While extracellular enzymes would be diluted if the protein of interest
directly gets released to the media, therefore would reduce the yield and need significant concentration
before being able to be used (Arbige, 2019).

#### References

* Klimentová, J., & Stulík, J. (2015). Methods of isolation and purification of outer membrane
  vesicles from gram-negative bacteria. Microbiological research, 170, 1-9.
* Arbige, M. V., Shetty, J. K., & Chotani, G. K. (2019). Industrial enzymology: the next chapter.
  Trends in biotechnology, 37(12), 1355-1366.
* Majumder, A., Purama, R. K., & Goyal, A. (2007). An overview of purification methods of glycoside
  hydrolase family 70 dextransucrase. Indian journal of microbiology, 47(3), 197-206.

[![](https://static.igem.org/mediawiki/2021/4/4c/T--UBrawijaya--Logo.png)](https://2021.igem.org/Team%3AUBrawijaya)

### Follow us

### Contact us

* igemub2021@gmail.com
* Jl. Veteran, Malang, East Java 65145

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

# Team:UBrawijaya/Experiments

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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

# Team:UBrawijaya/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)