iGEM 2019 — Team Saint_Joseph

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# Team:Saint Joseph/Notebook

Project Notebook

[![](https://static.igem.org/mediawiki/2019/8/81/T--Saint_Joseph--logo_250.png)](https://2019.igem.org/Team%3ASaint_Joseph)

* [Home](https://2019.igem.org/Team%3ASaint_Joseph)
* Project

  [Description](https://2019.igem.org/Team%3ASaint_Joseph/Description)
  [Design](https://2019.igem.org/Team%3ASaint_Joseph/Design)
  [Experiments](https://2019.igem.org/Team%3ASaint_Joseph/Experiments)
  [Results](https://2019.igem.org/Team%3ASaint_Joseph/Results)
  [Notebook](https://2019.igem.org/Team%3ASaint_Joseph/Notebook)
  [Protocols](https://2019.igem.org/Team%3ASaint_Joseph/Protocols)
  [Demonstrate](https://2019.igem.org/Team%3ASaint_Joseph/Demonstrate)
  [Future Work](https://2019.igem.org/Team%3ASaint_Joseph/Future)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ASaint_Joseph/Parts_Overview)
  [Basic Parts](https://2019.igem.org/Team%3ASaint_Joseph/Basic_Part)
  [Improved Parts](https://2019.igem.org/Team%3ASaint_Joseph/Improved_Parts)
  [Composite Parts](https://2019.igem.org/Team%3ASaint_Joseph/Composite_Parts)
* [Safety](https://2019.igem.org/Team%3ASaint_Joseph/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3ASaint_Joseph/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3ASaint_Joseph/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ASaint_Joseph/Collaborations)
* Team

  [Team Members](https://2019.igem.org/Team%3ASaint_Joseph/Team)
  [Attributions](https://2019.igem.org/Team%3ASaint_Joseph/Attributions)
  [Sponsors](https://2019.igem.org/Team%3ASaint_Joseph/Team#sponsors)
* [Gallery](https://2019.igem.org/Team%3ASaint_Joseph/Gallery)
* [Contact](#main-footer)
* [Judging Form ⇗](https://igem.org/2019_Judging_Form?team=Saint_Joseph)

# Notebook

![](https://static.igem.org/mediawiki/2019/e/ee/T--Saint_Joseph--dnamap.jpg)

# Day-1 : 07.07.2019

### SOC Medium Preparation

20mM glucose is added to the prepared 1L SOB to increase the
richness. The SOC medium is not autoclaved so that glucose is
not caramelized. The final pH is 6.8-7.

We're moving from LBA to LB Broth. Transition from solid medium
to liquid medium. While making this transition, we made a colony
selection, placed the colony we have taken on 10ml LB Broth
medium and left it for incubation.

### Creation of Competent Cells by CaCl2 Method

0.5 ml of overnight E.coli culture is added to 50 ml LB medium
and produced at 37 C at 180 rpm until OD600 = 0.3-0.4. The
culture is then allowed to stand on ice for 10 minutes. It is
then centrifuged at 4 ° C for 14 minutes at a speed of 14,000
rpm. Centrifugation is done to precipitate the cells. 10 ml of
0.1M CaCl 2 was added to the pellet and left on ice for 30
minutes. Centrifuged for 4 minutes at 14,000 rpm at 4 ° C. The
pellet is then suspended in 2 ml of 0.1 M CaCl 2, stored at 4 °
C. Finally, 200 microliters of cell suspension is transformed.

### E.Coli DH5a Bacteria Breed Transformation:

Added 1-10mL of DNA suspension containing 0.1-1 mg of DNA on 1
to 200 mL of sufficient cell suspension and allowed to stand for
30 minutes on ice. Then it is kept at 42 ° C for 90s and then
kept on ice for 2 minutes. 800 mL of LB or SOC medium (37 C) is
added and kept at 37 C for 30-90 minutes with gentle agitation.
And finally, 100mL / petri LBA selective medium is seeded.
Incubated for 1 night. Colony selection is made for the
transition from solid medium to liquid medium. We then left the
cells to incubate again for 1 day to proliferate in the liquid
medium.

# Day-2 : 16.07.2019

### LB Broth Medium

First we poured 25 gram lb Broth Powder to an autoclaved bottle
with screw cap. Then we completed it with 1 liter
ddH2O(distilled water). After that, we closed the lid halfly,
covered it with foil and glued autoclave tape on it. (The tape
which allows us to distinguish). Furthermore, we autoclaved it
at 121 degrees for 20 minutes. Lastly, after letting it to cool
to room temperature, we kept it at 4 degrees.

### LB Agar Medium Making

We prepared 1 liter LB liquid medium in a 2 liter erlenmeyer.
Then, we added 15 grams of bacterial agar and we blended it. We
autoclaved it at 121 degrees for 15 minutes and cooled it to 50
degrees. We added antibiotics and blended them by spining the
bottle strongly. We label the antibiotic from its inside. Lastly
we poured the medium into petri plates but while doing it we
paid attention that it doesn’t cool down because agar gets
polymerised when it cools under 60 degrees. In addition, while
adding the antibiotic, we avoided the temperature getting high.

### LBA Medium Plantation

We took the medium with a 1- 0,1= 100 microliter pipette and put
it in the middle of the petri plates. With the help of an
alcohol sterilised and singed baguette we finished the process
of propagation.

### SOB Preparation

It gets disintegrated in 0,5g NaCl and 20g Tripton 950 ml dH2O.
We added 5g Yeast Extract. Yeast Extract is a very rich medium
just like a yeast medium. We prepared a proper environment for
the bacterias. Then we shaked it until it gets dissolved and we
added 10 mL of 250 mM KCL stock. Later, we adjusted the mediums
pH to 7 by adding 5g NaOH. We fulfiled the solution with dH2O to
1 liter. We sterilised it by autoclaving 15 to 20 minutes in
psi. Before using it, sterilised 2M MgCl2 should be added.( It
should be absolutely added after the autoclaving step)

# Day-3 : 19.07.2019

### Agarose gel electrophoresis

To prepare agarose gel, we mixed 0.5 g of agarose with 75 ml of
tae buffer. We heated the mixture three times in the microwave
for one minute and stirred. We added a drop of ethidium bromure
to the cassette in the dark (to preserve its dyeing ability),
and when the agarose gel we prepared cooled down, we added it to
the cassette. After waiting for half an hour for the gel to
polymerize, we put the cassette in the tank, added the
sterilized water, and then we added:

1. Pet26
2. Laccase
3. Uncut pet26
4. Uncut laccase.

We waited for 25 minutes for the DNA to walk.

Then we compared them in UV light.

### Ligation

We mixed 4 microliters of buffer, 1 microliter of enzyme, 7
microliter of pet26, 3 microliter of laccase and 5 microliter of
nuclease free water. And we stood at 16 degrees. Then we
transformed them and checked them.

### High Efficiency Transformation

We transferred gene to the bacteria because we want to
reproducing the bacteria

Protocol

1. Thaw a tube of SHuffle Competent E. coli cells on ice for 10
   minutes
2. Add 1–5 μl containing 1 pg–100 ng of plasmid DNA to the cell
   mixture. Carefully flick the tube 4–5 times to mix cells and
   DNA. Do not vortex
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 μl of room temperature SOC into the mixture.
7. Place at 30°C for 60 minutes. Shake vigorously (250 rpm) or
   rotate.
8. Warm selection plates to 30°C.
9. Mix the cells thoroughly by flicking the tube and inverting,
   then perform several fold serial dilutions in SOC.
10. Spread 50–100 μl of each dilution onto a selection plate and
    incubate overnight at 30°C. Alternatively, incubate at 25°C
    for 48 hours.

### Expression Using SHuffle

We made gene expression for turning gene to the protein and for
producing enzyme.

Protocol

1. Transform expression plasmid into SHuffle. Plate on antibiotic
   selection plates and incubate 24 hours at 30°C
2. Resuspend a single colony in 10 ml liquid medium with
   antibiotic.
3. Incubate at 30°C until OD600 reaches 0.4–0.8.
4. Add the appropriate inducer, e.g. 40 μl of a 100 mM stock of
   IPTG. Incubate for 4 hours at 30°C or 16°C
5. Check for expression either by Coomassie stained protein gel,
   Western Blot or activity assay. Check expression in both the
   total cell extract (soluble + insoluble) and the soluble
   fraction alone.
6. For large scale, innoculate 1 L of liquid medium (with
   antibiotic) with a freshly grown colony or 10 ml of freshly
   grown culture. Incubate at 30°C until reaches 0.4–0.8. Add the
   appropriate inducer, e.g. IPTG to 0.4 mM. Induce 4 hours or
   16°C overnight.

### Protein Isolation

E.coli SHuffle cells including the recombinant laccase gene were
induced by IPTG overnight for expression of laccase enzymes.

After expression, polyhistidine tagged laccase enzymes were
isolated with magnetic tube rack (GeneOn) and the MagneHis
Protein Purification System (Promega).

### Restriction Enzyme Digestion

We added restriction sites on the two ends of the laccase gene,
which are necessary for the enzymes to cut the DNA and suitable
for the expression vector.

While selecting the enzymes we were going to use, we paid
attention that recognition sites of the enzymes are not within
the laccase gene. In this way, we prevented the enzymes from
cutting the gene and causing the gene's inability to function.

We used two different restriction enzymes to prevent the cut
ends from being complementary and sticking to themselves.

We isolated the expression vector and laccase gene from cut
products analyzed by agarose gel electrophoresis. After the
density of the DNA extracted from the gel was determined, we
ligated the vector and the gene to be cloned in a 3:1 ratio.

### The Protocol

Restriction Enzyme...............1 μl
DNA..............................1 μg
10X NEBuffer.....................5 μl (1X)
Total Rxn Volume.................50 μl
Incubation Temperature...........37 C
Incubation Time..................1 hour

# Day 4 : 20.07.2019

### Plasmid Isolation Protocol with Kit

1. Collection of bacteria: E.coli in 1.5 ml micro-centrifuge
   pellet is taken to overnight culture in 1.5-5 ml LB medium
   containing appropriate antibiotic.
Note:
Solution 1,2,3 allows cell membrane disintegration because it
contains SDS, it breaks down the lipids in the cell membrane.
It removes the proteins.2. Resuspension: We added 100 micro-liters of “solution 1 ”and we
   resuspended the bacterial palette with vortex.
3. Lysis: We added 200 micro-liters of “solution 2”, and mixed
   slowly by inverting the micro-tubes 4-5 times, keeping it at
   room temperature for 1 minute. If “VisualLyse” is added, the
   solution turns blue, stirring slowly must be done until a
   homogeneous blue solution is obtained.
4. Neutralization: We added 350 micro-liters of ‘’solution 3’’.
   We slowly mixed the micro-tube 4-6 times and let it stand at
   room temperature for 1 minute. If ‘’VisualLyse ”is added,
   mixing slowly must be done until all blue marks are removed.
5. Pellet Cell residues are centrifuged for 5 minutes at 10000
   rpm in micro-centrifuge.
6. DNA binding is transferred to the supernatant spin column. It
   is then centrifuged for 2 minutes and the flowing liquid
   discarded. Special bit tube with supernatant spin column
   filter.
7. Washing: 750 micro-liters of Solution Wash Solution is added
   and centrifuged for 1 minute and then the flowing liquid is
   discarded.
8. Wash the Column again.
9. Centrifuge for 1 minute to remove any “Wash Solution” residue.
10. Elution: Transfer the column to a new 1.5 ml micro-tube. Add
    50 micro-liters of Elution Buffer to the center of the Spin
    Column and centrifuge at 10,000 rpm for 2 minutes. The elution
    buffer washes the plasmid DNA in the filter.

# Day 5 : 25.07.2019

### Recovery from gel

(We will look at the gel to make sure there is a cut)

Weigh the gel (0.24 g) (we put 1000 times the amount of gel in
it)

We thawed the gel in 240 microliters of DB buffer.

Spined the gel. The gel settled.

We melted the gel at 50 ° C.

We transferred the melted gel solution to the column and
centrifuged at 10000 rpm for 1 minute and discarded the
residue. (To get rid of DNA waste)

We washed the column with 650 milliliters of wash buffer.

We centrifuged it at 10000 rpm for 1 minute.

As a guarantee, we washed the column again with wash buffer
and centrifuged again at 10000 rpm for 1 minute.

We centrifuged it at 10000rpm for 1 minute without adding
buffer.

To precipitate the DNA we binded it to the column, we first
added 30 microliters of nuclease free water to the column and
then centrifuged it, then added 20 microliters and centrifuged
it again (50 microliters in total). And we raised it to -20
degrees.

---

After cloning

We've isolated it to increase our DNA stock.

After expression

We ligated the DNA which came to us. Because that we ligate in
small amounts, we cannot put them directly on the gel and
control them. We cloned and amplified DNA into good bacteria,
and isolated it. We first isolated it to check whether the
laccase and the expression vector were correctly bounded and
to check whether the laccase gene was cloned into this
expression vector. We loaded the plasmid DNA on our gel
without ever cutting it. We applied the cutting enzymes we
used here to isolate the material and wait for it to cut. To
control this, we isolated the DNA after expression and looked
at the DNA in gel. We stocked some of our isolates and used
them for control.

Purification with gel

In order to remove the useless part of the plasmid that came
to us and to remove the unwanted parts in the vector, we first
electrophoresed and separated these parts and also checked
whether our segments were correct. If our cut is correct, we
took the small part corresponding to the laccase and the big
part corresponding to the expression vector, isolating them
from the gel and connecting them together. Thus, the laccase
was in the ve expression bacteria we wanted.

### Procedure

After Expression

Before using, heat the lisis and neutralisation buffers at
37°C then wait for them to cool down at 25°C. Heat the elution
buffer to 70 ° C and apply to column.

Collecting bacterias

Collect bacterial culture at 8000rpm (6800 x g) for 2
minutes in a centrifuge.

Carefully empty the supernatant and remove any residue.

Suspended cells, thawing, neutralization

To pellet cellules:

* Add 250µL Resuspension Solution and vortex
* Add 250µL Lysis Solution and turn the tube inside 4-6
  times
* Add 350µL Neutralization Solution and turn the tube
  inside 4-6 times

Centrifuge for 5 minutes

Linking DNA

Transfer supernatant to Thermo Scientific GeneJET Spin
Column

Washing the column

Add 500µL Wash Solution and centrifuge for 30-60 seconds,
then empty the liquid. ( x 2 )

Centrifuge the empty column for 1 minute.

DNA recovery from agarose ge

Gel electrophoresis

Run DNA Sample on agarose gel electrophoresis to seperate
DNA fragments. Perform ethidium bromide staining for DNA
visualization.

Cut agarose gel band containing the desired DNA and place
it into a pre-weighed microcentrifuge tube.

Solubilization of agarose

Determine the nett weight of gel slice and add 1 volume of
Buffer DB to 1 volume of gel.

Centrifuge the tube briefly to make sure the gel slice
stays at the bottom of the tube.

Incubate at 50°C until gel has melted completely.

Mix occasionally to ensure complete solubilisation.

DNA purification

Loading to column

Transfer the sample into a column assembled in a clean
collection tube. Centrifuge at 10,000 x g for 1 min.
Discard flow through.

Repeat for any remaining sample from step 2.

Column washing

DWash the column with 650µl wash buffer and centrifuge at
10,000 x g for 1 min. Discard flow through.

Column drying

Centrifuge the column at 10,000 x g for 1 min to remove
residual ethanol.

### iGEM Team Saint Joseph

Follow Us

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# Team:Saint Joseph/Experiments

Experiments

[![](https://static.igem.org/mediawiki/2019/8/81/T--Saint_Joseph--logo_250.png)](https://2019.igem.org/Team%3ASaint_Joseph)

* [Home](https://2019.igem.org/Team%3ASaint_Joseph)
* Project

  [Description](https://2019.igem.org/Team%3ASaint_Joseph/Description)
  [Design](https://2019.igem.org/Team%3ASaint_Joseph/Design)
  [Experiments](https://2019.igem.org/Team%3ASaint_Joseph/Experiments)
  [Results](https://2019.igem.org/Team%3ASaint_Joseph/Results)
  [Notebook](https://2019.igem.org/Team%3ASaint_Joseph/Notebook)
  [Protocols](https://2019.igem.org/Team%3ASaint_Joseph/Protocols)
  [Demonstrate](https://2019.igem.org/Team%3ASaint_Joseph/Demonstrate)
  [Future Work](https://2019.igem.org/Team%3ASaint_Joseph/Future)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ASaint_Joseph/Parts_Overview)
  [Basic Parts](https://2019.igem.org/Team%3ASaint_Joseph/Basic_Part)
  [Improved Parts](https://2019.igem.org/Team%3ASaint_Joseph/Improved_Parts)
  [Composite Parts](https://2019.igem.org/Team%3ASaint_Joseph/Composite_Parts)
* [Safety](https://2019.igem.org/Team%3ASaint_Joseph/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3ASaint_Joseph/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3ASaint_Joseph/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ASaint_Joseph/Collaborations)
* Team

  [Team Members](https://2019.igem.org/Team%3ASaint_Joseph/Team)
  [Attributions](https://2019.igem.org/Team%3ASaint_Joseph/Attributions)
  [Sponsors](https://2019.igem.org/Team%3ASaint_Joseph/Team#sponsors)
* [Gallery](https://2019.igem.org/Team%3ASaint_Joseph/Gallery)
* [Contact](#main-footer)
* [Judging Form ⇗](https://igem.org/2019_Judging_Form?team=Saint_Joseph)

# Experiments

# Strategy

[![SJT](https://static.igem.org/mediawiki/2019/d/df/T--Saint_Joseph--ex-sum.jpg)](https://static.igem.org/mediawiki/2019/d/df/T--Saint_Joseph--ex-sum.jpg)

Provide the laccase enzyme to be directed to periplasmic area
and increase the total amount of purified enzyme from E. coli.

[![SJT](https://static.igem.org/mediawiki/2019/7/74/T--Saint_Joseph--exp_01.jpg)](https://static.igem.org/mediawiki/2019/7/74/T--Saint_Joseph--exp_01.jpg)

Laccase is an enzyme which cleans textile waste water by
decolorization. We used two methods: We designed the laccase
enzyme produced by a white-rot fungi called Trametes versicolor
bioinformatically.

[![SJT](https://static.igem.org/mediawiki/2019/8/80/T--Saint_Joseph--exp_07.jpg)](https://static.igem.org/mediawiki/2019/8/80/T--Saint_Joseph--exp_07.jpg)

### Why Trametes versicolor?

Trametes versicolor is a white-rot fungi. Laccase has
multiple different sources but studies show that laccase
produced by Trametes versicolor has wide-range usage area.
The activity of enzyme is higher than the laccases produced
from other organisms

[![SJT](https://static.igem.org/mediawiki/2019/3/30/T--Saint_Joseph--exp_02.jpg)](https://static.igem.org/mediawiki/2019/3/30/T--Saint_Joseph--exp_02.jpg)

### Why DSB region?

PelB sequence is added to investigate if the activity of laccase
increases when the enzyme is secreted to the periplasmic region.

### Why PelB signal sequence?

PelB sequence is added to investigate if the activity of laccase
increases when the enzyme is secreted to the periplasmic region.

### Why *E.coli* SHuffle Strain?

*E.coli* SHuffle strain promotes disulfide bond formation
in cytoplasm. Disulfide bonds increase the activity of the
enzymes.

# Methodology

### Bioinformatic Design

Plasmids used in the studies are designed with Benchling
programme

### Recombinant DNA Construction

Cloning vector and laccase gene are transformed into
*E.coli* DH5α strains and produced separately. After
production they are isolated and cut with restriction enzymes.
Cloning vector and laccase gene are ligated and finally laccase
gene is cloned.

### Protein Expression

Cloned laccase genes are isolated and then transformed into
expression vector to obtain protein. After expression step,
proteins are isolated.

* 1. WILD TYPE laccase

  We optimised it because we want to avoid codon
  degeneration. We cloned this enzyme in *E.coli* DH5α
  and we produced it in *E.coli* SHuffle strain. Once
  the enzyme got produced in SHuffle, we isolated the enzyme
  and tested it.

  [![SJT](https://static.igem.org/mediawiki/2019/c/c4/T--Saint_Joseph--desc_4.jpg)](https://static.igem.org/mediawiki/2019/c/c4/T--Saint_Joseph--desc_4.jpg)
* 2. pelB + LACCASE

  We added pelB signal sequence to laccase gene, and we
  requested our designed sequence in a cloning plasmid from
  IDT.

  [![SJT](https://static.igem.org/mediawiki/2019/9/9c/T--Saint_Joseph--exp_04.jpg)](https://static.igem.org/mediawiki/2019/9/9c/T--Saint_Joseph--exp_04.jpg)

  We transformed this plasmid to E. coli DH5α for cloning
  the sequence. We transferred cloned sequences to SHuffle
  strain for expression of the laccase enzyme. The enzyme is
  secreted from the cytoplasm to the periplasm.
* 3. DSB + LACCASE

  We added the disulphide bonding isomerase (DSB) sequence
  to laccase gene, and we requested our designed sequence in
  a cloning plasmid from IDT.

  [![SJT](https://static.igem.org/mediawiki/2019/e/e1/T--Saint_Joseph--exp_03.jpg)](https://static.igem.org/mediawiki/2019/e/e1/T--Saint_Joseph--exp_03.jpg)

  We transformed this plasmid to E. coli DH5α for cloning
  the sequence. We transferred cloned sequences to SHuffle
  strain for expression of the laccase enzyme. DSB region is
  added to wild-type laccase for increasing disulphide bond
  formation and enzyme activity.

# Our Plasmid

[![SJT](https://static.igem.org/mediawiki/2019/c/ca/T--Saint_Joseph--exp_05.jpg)](https://static.igem.org/mediawiki/2019/c/ca/T--Saint_Joseph--exp_05.jpg)

* *E.Coli* SHuffle

  The laccase enzyme has disulfide bonds in its wild type
  form. Laccase with these disulfide bonds cannot be produced
  in *E.Coli* DH5α. That is why we transformed it to the
  SHuffle strain to produce the laccase enzyme. This strain
  can form disulfide bonds of the protein naturally so we do
  not need to induce disulfide bond formation.
  *E.Coli* DH5α is only used for cloning.
* Isolation of Laccase

  The designed laccase enzymes has histidine tags at the end.
  The isolation kit we got contains nickel particles. These
  particles recognize and bind to histidine tags. We placed
  this mixture on a magnetic tube stand. Enzymes stick to the
  wall with the magnet and the remaining liquid, the
  supernatant, is discarded. The remained pellet is our
  enzyme.

  After isolating the enzyme produced in SHuffle , we stored
  it at -20 oC. Then we interacted these enzymes
  with dyed water.

# Measurement Kit

We did the measurement experiment with iGEM Kit.

[![SJT](https://static.igem.org/mediawiki/2019/7/71/T--Saint_Joseph--measure-kit.jpg)](https://static.igem.org/mediawiki/2019/7/71/T--Saint_Joseph--measure-kit.jpg)

##### Measurement Kit

### Plasmid Isolation Protocol with Kit

* 1- Collection of bacteria: E.coli in 1.5 ml
  micro-centrifuge pellet is taken to overnight culture in
  1.5-5 ml LB medium containing appropriate antibiotic.

  Note: Solution 1,2,3 allows cell membrane disintegration
  because it contains SDS, it breaks down the lipids in the
  cell membrane. It removes the proteins.
* 2- Resuspension: We added 100 micro-liters of “solution 1
  ”and we resuspended the bacterial palette with vortex.
* 3-Lysis: We added 200 micro-liters of “solution 2”, and
  mixed slowly by inverting the micro-tubes 4-5 times,
  keeping it at room temperature for 1 minute. If
  “VisualLyse” is added, the solution turns blue, stirring
  slowly until a homogeneous blue solution is obtained.
* 4-Neutralization: We added 350 micro-liters of solution3.
  We slowly mix the micro-tube 4-6 times and let stand at
  room temperature for 1 minute. If Ly Visual Lyse ”is
  added, mix slowly until all blue marks are removed.
* 5- Pellet Cell residues are centrifuged for 5 minutes at
  10000 rpm in micro-centrifuge.
* 6. DNA binding is transferred to the supernatant spin
  column. It is then centrifuged for 2 minutes and the
  flowing liquid discarded. Special bit tube with
  supernatant spin column filter.
* 7- Washing: 750 micro-liters of Solution Wash Solution ”is
  added and centrifuged for 1 minute and then the flowing
  liquid is discarded.
* 8- Wash the Column again.
* 9. Centrifuge for 1 minute to remove any “Wash Solution”
  residue.
* 10- Elution: Transfer the column to a new 1.5 ml
  micro-tube. Add 50 micro-liters of Elution Buffer to the
  center of the Spin Column and centrifuge at 10,000 rpm for
  2 minutes. The elution buffer washes the plasmid DNA in
  the filter.

### iGEM Team Saint Joseph

Follow Us

Copyright ©

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Saint Joseph/Results

Results

[![](https://static.igem.org/mediawiki/2019/8/81/T--Saint_Joseph--logo_250.png)](https://2019.igem.org/Team%3ASaint_Joseph)

* [Home](https://2019.igem.org/Team%3ASaint_Joseph)
* Project

  [Description](https://2019.igem.org/Team%3ASaint_Joseph/Description)
  [Design](https://2019.igem.org/Team%3ASaint_Joseph/Design)
  [Experiments](https://2019.igem.org/Team%3ASaint_Joseph/Experiments)
  [Results](https://2019.igem.org/Team%3ASaint_Joseph/Results)
  [Notebook](https://2019.igem.org/Team%3ASaint_Joseph/Notebook)
  [Protocols](https://2019.igem.org/Team%3ASaint_Joseph/Protocols)
  [Demonstrate](https://2019.igem.org/Team%3ASaint_Joseph/Demonstrate)
  [Future Work](https://2019.igem.org/Team%3ASaint_Joseph/Future)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ASaint_Joseph/Parts_Overview)
  [Basic Parts](https://2019.igem.org/Team%3ASaint_Joseph/Basic_Part)
  [Improved Parts](https://2019.igem.org/Team%3ASaint_Joseph/Improved_Parts)
  [Composite Parts](https://2019.igem.org/Team%3ASaint_Joseph/Composite_Parts)
* [Safety](https://2019.igem.org/Team%3ASaint_Joseph/Safety)
* Human Practices

  [Integrated Human Practices](https://2019.igem.org/Team%3ASaint_Joseph/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3ASaint_Joseph/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ASaint_Joseph/Collaborations)
* Team

  [Team Members](https://2019.igem.org/Team%3ASaint_Joseph/Team)
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# RESULTS

# In silico Design

* The laccase gene was codon optimized for expression in
  E.coli.
* The laccase gene and pET26b expression vector were analyzed
  and appropriate restriction endonucleases were selected.

* Recombinant DNA was modeled by simulating digestion and
  ligation processes with restriction enzyme on Benchling.
* Each of these processes was performed separately for
  wild-type laccase, pelB.laccase and dsb.laccase

# Plasmid Amplification by Transformation

The pUC cloning plasmids carrying the laccase gene (wild-type,
dsb.laccase and pelB.laccase) were transformed into E. coli DH5α
cells by heat shock method and colony selection performed on
ampicillin agar plates.

[![plasmid](https://static.igem.org/mediawiki/2019/f/ff/T--Saint_Joseph--plasmid_01.jpg)](https://static.igem.org/mediawiki/2019/f/ff/T--Saint_Joseph--plasmid_01.jpg)

[![plasmid](https://static.igem.org/mediawiki/2019/3/36/T--Saint_Joseph--plasmid_02.jpg)](https://static.igem.org/mediawiki/2019/3/36/T--Saint_Joseph--plasmid_02.jpg)

[![plasmid](https://static.igem.org/mediawiki/2019/3/38/T--Saint_Joseph--plasmid_03.jpg)](https://static.igem.org/mediawiki/2019/3/38/T--Saint_Joseph--plasmid_03.jpg)

# Plasmid Isolation

* Plasmids cloned into E.coli DH5α cells were isolated with
  the ABM Column-Pure Plasmid Miniprep Kit. The VisualLyse
  indicator was used to fully understand that bacterial lysis
  occurred.
* The isolation results were measured with nanodrop
  (Thermofisher).

  ![plasmid](https://static.igem.org/mediawiki/2019/3/31/T--Saint_Joseph--plasmid_04.jpg)

# Cutting with Restriction Endonucleases

The pUC plasmids containing the laccase gene and the pET26b
expression vector were with HindIII and NdeI enzymes..

* The digested pET26b and laccase genes isolated from the gel.
  Then, isolated fragments were measured on the nanodrop and
  checked on the gel.
* Since another band anormally was imaged on the gel and it
  was difficult to distinguish with the help of ladder due to
  the proximity of the bands, both bands continued to work
  until sequencing results were obtained. These fragments are
  called heavy and light.

  | Sample ID | Nucleic Acid Unit | A260(Abs) | A280(Abs) | 260/280 | 260/230 |
  | --- | --- | --- | --- | --- | --- |
  | dpET26b 1 | 50.4 ng/µl | 1.008 | 0.719 | 1.4 | 0.27 |
  | dpET26b 2 | 0.8 ng/µl | 1.016 | -0.041 | -0.39 | 0 |
  | dsb light | 26.1 ng/µl | 0,521 | 0.242 | 2.15 | 0.08 |
  | dsb heavy | 48 ng/µl | 0.94 | 0.547 | 1.72 | 0.15 |
  | wild-type light | 47.4 ng/µl | 0.947 | 0.585 | 1.62 | 0.12 |
  | wild-type heavy | 32.9 ng/µl | 0.65 | 0.297 | 2.22 | 0.06 |
  | pelB light | 8.9 ng/µl | 0.179 | 0.048 | 3.75 | 0.01 |
  | pelB heavy | 8.9 ng/µl | 0.177 | 0.047 | 3.77 | 0.02 |

# Ligation

The DNA fragments isolated from the gel after digestion were
ligated with T4 DNA Ligase enzyme.

Recombinant DNA cloned into E.coli DH5 cells for amplifiying,
was isolated and transformed into E.coli SHuffle cells for
expression.

* After transformation, transformant colonies were selected on
  LBA medium containing antibiotics.

# Protein Isolation

E.coli SHuffle cells including the recombinant laccase gene were
induced by IPTG overnight for expression of laccase enzymes

After expression, polyhistidine tagged laccase enzymes were
isolated with magnetic tube rack (GeneOn) and the MagneHis
Protein Purification System (Promega).

![protein](https://static.igem.org/mediawiki/2019/a/a6/T--Saint_Joseph--protein_01.jpg)

### iGEM Team Saint Joseph

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