iGEM 2023 — Team cnas-romania

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## Notebook

Tracking your actions is the compass to your progress.

[See our notebook](#notebook)

* Month of January — We obtained sponsorship to cover the registration fee.
* 20th of April — We signed up to be a team in the iGEM.
* 29th of April – We received the confirmation that we were accepted.
* 8th of May — We signed up for the mentorship program offered by iGEM.
* 25th of May — We had our first meeting with the sweetest mentor ever.
* 30th of May — We received confirmation that we will be awarded a grant from the Ministry of Research, Innovation and Digitalisation that can cover all the fees left.
* 16th of June — We began working on the wiki.
* 22nd of June — We turned in the Safety Form.
* 26th of June — We uploaded our project description on our wiki.
* Beginning of July — We ordered the necessary materials for our experimental part.
* 16th of July — We wrote a cloning protocol for our first experiment.
* 31st of July — We had to change the protocol because the restriction enzyme BsaI wasn’t the right fit for what we wanted to do. (More explained [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D))
* 1st of August — We modified our IDT order and our cloning protocol again.
* 4th of August — We had a meeting at the university. The cyanobacteria medium BG-11 arrived.
* 5th of August — We moved the cyanobacteria into their growth medium BG-11.
* 6th of August — We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 7th of August — We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 8th of August — We modified the cloning protocol. We went to the university. We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 9th of August — We modified the cloning protocol. (More explained [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)) We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 10th of August — We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 11th of August — Online meeting with our teacher Mr. Badea, our mentor, and the team to discuss the cloning protocol. We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 12th of August — We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 13th of August — We modified the cloning protocol, adding another step. We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 14th-16th of August — We went to the lab to check on our cyanobacteria and perform the daily experiments.
* 17th of August-14th of September — We worked on interpreting data from our experiments, uploaded content for the wiki.
* 7th of September — We attended the “Life Safe Crossing” Conference.
* 15th of September — We had a meeting with one of our teachers. After reviewing everything we wrote up until that point, we decided we had to redo one of the experiments to be sure we got all the right data.
* 16th-25th of September — We worked on the deliverables for our medal criteria and uploaded content of the wiki.
* 6th-12th of October — We reviewed with our teachers what we had written so far and modified where we needed to. We finished uploading content of the wiki as well as our contributions to the Registry.

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## Engineering

In molecular biology, precision in experimental design empowers profound discoveries.

[See our work](#Cycle1)

* [Overview](#overview)
* [Cycle 1](#Cycle1)

  + [Design #1](#design1)
  + [Build and test #1](#build&test1)
  + [Learn #1](#learn1)
* [Cycle 2](#Cycle2)

  + [Design #2](#design2)
  + [Build and test #2](#build&test2)
  + [Learn #2](#learn2)

# Overview

In molecular biology there are many unknowns that can appear during an
experiment, which is why the correct construction of an experimental design is particularly important.
Before working starting wetlab work, we followed 2 engineering cycles that helped to refine the
construct that we intended to create. It is important to mention that, in our case, the build and test
stages represent a modelling phase, through which the drawn conclusions led to the learning stage.

## First Cycle

## Design #1

We started documentation for the properties of alkB1, the main gene of
interest. Another iGEM team(TzuChiU\_Formosa) created a part containg alkB1 using RBS BBa\_B0034,
however we chose to use RBS BBa\_B0032 instead because it has higher strength which results in higher
protein synthesis. The idea then was to insert alkB1 with BBa\_B0032 to have a continuous expression
and degradation of present alkanes. As for the plasmid backbone we decided to use plasmid pSB1C5A from
the distribution kit.

As only cloning and observing the permanent expression of alkB1 would not have been very efficient and
might have led to an unsatisfactory result, we decided to use alkS as it is the transcription factor
for palkB, which will activate the expression of alkB1, being the inducible promoter for this gene.

![](https://static.igem.wiki/teams/4740/wiki/fig1e.png)

*Figure 1: alkane degradation genes mechanism*

The strategy was to take a plasmid with a constitutive promoter from the distribution
kit and clone alkS in that plasmid. Then, using another plasmid from the distribution kit, clone palkB and
alkB1 into it, with palkB placed upstream of alkB1, and then do a three-point ligation to insert both genes
into the open plasmid. After that, the plan was to transform both plasmids into the same E. coli cells, so
the alkS-palkB-alkB1 complex works.
We chose to use the J23119 promoter for the alkS gene and clone both of them into pSB1C30 from the
distribution kit (cut pSB1C30 and plasmid containing J23119 with BsaI and then ligate them, cut alkS with
XbaI and PstI, cut pSB1C30-J23119 with SpeI and PstI and ligate alkS into open pSB1C30-J23119). For palkB
and alkB1, we chose to use the pJUMP28-1A (sfGFP) containing BBa\_J428353 as backbone (Kanamycin resistance)
and clone both genes into the plasmid.

## Build and Test #1

After looking into the BsaI enzyme, we decided it is not correct to use it as it cuts
4 bases away from the recognition sequence, therefore not creating compatible ends. This restriction enzyme
is an IIS-type enzyme, not the IIP-type as the other ones, which means it cuts the DNA after the palindromic
region that the enzyme recognizes, not in the recognition site.
Thus, if we cut with this enzyme, we would obtain from the pSB1C30 plasmid the sequences found in the images
below.

![](https://static.igem.wiki/teams/4740/wiki/fig2a-e.png)

![](https://static.igem.wiki/teams/4740/wiki/fig2b-e.png)

*Figures 2: BsaI restriction enzyme cutting for plasmid pSB1C30*

From the plasmid containing the J23119 promoter we obtained the sequences found in the
images below:

![](https://static.igem.wiki/teams/4740/wiki/fig3a-e.png)

![](https://static.igem.wiki/teams/4740/wiki/fig3b-e.png)

*Figures 3: BsaI restriction enzyme cutting for promoter J20119*

Although there is nothing wrong with the cutting process itself, a problem arises when the piece of DNA
containing the promoter is inserted into the pSB1C30 plasmid, because the overhangs that remain are not
compatible with each other. For example, 5' GCTT 3' in figure 2b should be compatible with 3' GGAG 5' in
figure 3b, which is not true.
A solution to solve this problem would be to use PCR, with primers that amplify the promoter and add
additional base pairs to be cut with BsaI so that ends are compatible.

## Learn #1

The solution we chose however is to cut the pSB1C30 backbone with EcorI and PstI,
order the J23119 promoter sequence separately and cut it with EcorI and SpeI, and then do a three-point
ligation with the open plasmid, the cut J23119, and the alkS cut with XbaI and PstI.

## Second Cycle

## Design #2

Identified problem: Both the pSB1C30 and the pJUMP28-1A have the pMB1 origin,
therefore making them incompatible to transform in the same cell. We also added “ggc” overhangs at the ends
of the sequences which used enzymes that cut at the end of the sequence in order to make sure we have stable
compatible ends.

pJUMP28-1A was replaced with pJUMP26-1A, which has a p15A origin with a medium copy number, therefore making
the plasmids compatible. We decided to place the J23119-alkS complex in pJUMP26-1A and the palkB-alkB1
complex in pSB1C30, as pSB1C30 has a high copy number, hence we thought it would be better to use it to
ensure the noticeable expression of alkB1.

## Build and Test #2

The palkB sequence we were planning to order was incorrect, as the sequence we were
using was supposed to be cloned upstream of alkB1, so we reversed the sequence, without changing the
direction of the prefix and the suffix, so the promoter would transcribe in the right direction. We also
replaced the EcorI recognition sites from the alkB1 sequence to prevent alteration of open reading frames by
modifying codon nucleotides, without changing the corresponding amino acid sequence.

We realized it would be less complicated and we would have a higher chance of success if we cloned all 4
sequences of interest (J23119 promoter, alkS, palkB promoter and alkB1) into the same plasmid (pSB1C30, as
it has a high copy number), with the J23119-alkS complex cloned upstream of the palkB-alkB1 complex, in
order to minimize leakiness and problems derived from inefficient transcription termination or translation
readthrough. After having both plasmids with their corresponding gene complexes inserted, we would cut
J23119-alkS out of pJUMP26-1A using XbaI and PstI and ligate it with pSB1C30 cut with SpeI and PstI,
therefore obtaining a bicistronic plasmid

## Learn #2

As a result of all iterations, the final construct to be created was modeled in SnapGene.

![](https://static.igem.wiki/teams/4740/wiki/fig4e.png)

*Figure 4: plasmid construct with J23119, alkS, palkB and alkB1*

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## Experiments

Experiments are the engines of discovery, igniting the flames of discovery.

[See our work](#expd)

* [Overview](#overview)
* [Experiments description](#expd)
* [Cloning protocol](#cloningprotocol)
* [Standard protocols](#standardprotocols)
* [References](#references)

# Overview

In our project, we developed an alkane sensing part that degrades oil found in
water. However, the experiments proposed were not finished entirely. There are several possibilities
to study this project in the future by introducing the recombinant plasmids into the targeted
organisms, cyanobacteria, through electroporation. Later, these organisms containing the genes of
interest will be tested in a mixture containing alkanes.

## 1.Experiments description

## 1.1.Preparation of LB Broth

To begin the cloning experiment, we first prepared LB Broth. This required mixing 25
grams of LB Broth powder (Scharlau, cat# 02-385) with every 1 litre of distilled water. After thorough
mixing, we autoclaved the solution at 121°C for 15 minutes, using a total volume of 0.5 litres.
Subsequently, we completed the mixture by adding autoclaved water to reach the 1-litre mark.

![](https://static.igem.wiki/teams/4740/wiki/zfig1exp.jpg)

*Fig.1-LB broth liquid medium*

## 1.2.Preparation of LB Agar

LB Agar was prepared by dissolving 20 grams of LB Agar powder (Scharlau, cat# 01-385)
in 0.5 litres of distilled water. After heating the mixture to melt the agar, it was autoclaved at 121°C for
15 minutes. 25 μl of the agar solution were then poured onto each agar plate, and these plates were allowed
to cool down.

## 1.3.Preparation of 1 M CaCl2 Solution

Our next step involved preparing a 1 M CaCl2 solution. Using CaCl2 anhydrous (C1016)
with a molecular weight of 110.98, we calculated that 200 millilitres of the solution required approximately
22.2 grams of CaCl2. We mixed the calculated amount of CaCl2 with distilled water to obtain the desired 1 M
CaCl2 solution.

## 1.4.Preparation of Competent Cells

On August 24, 2023, we initiated the preparation of competent E. coli cells for transformation. We began by
plating 200 microliters, sourced from a glycerol stock, onto two plates, designating them as "DH5 alpha 1"
and "DH5 alpha 2." These plates were specifically chosen for their capacity to support nearly confluent cell
growth. The plates were subsequently stored at 4°C on August 25, 2023. On August 27, 2023, we performed
colony selection by picking three colonies from each plate and placing them into 2 ml glass tubes. These
tubes were then subjected to overnight shaking at 320 RPM and 37°C.

![](https://static.igem.wiki/teams/4740/wiki/zfig2exp.png)

*Fig.2- E.coli colonies on agar plate*

On August 28, 2023, we transferred 250 μl from the overnight culture into 50 ml flasks, aiming to grow the
culture until the optical density at 600 nm (O.D.600) reached the desired range of 0.4 - 0.6. Utilising a
densitometer for monitoring, we noted that the overnight culture had a reading of 1.4. After 2:45 hours
after the start of the growth, the density stood at 3, while at 3:30 hours, it had reached 5.1, leading us
to halt the growth process.

Following growth, we centrifuged the cells at 5000 g for 5 minutes to obtain pellets. The pellets were
resuspended in 25 ml of 0.1 M CaCl2, prepared using 2.5 ml of 1 M CaCl2. After an hour of incubation on ice,
with a brief benchtop rest for 5 minutes, the suspension was subjected to a second centrifugation step at
5000 g for 5 minutes. Following the second centrifugation, the pellet was resuspended in 3 ml of a chilled
solution consisting of 15% Glycerol and 0.1 M CaCl2. This solution was prepared by mixing 450 μl of
glycerol, 300 μl of 1 M CaCl2, and 2.25 ml of H2O. Finally, we aliquoted 100 μl of the suspension and stored
it at -80°C for future use.

## 1.5.Transformation of DH5 alpha Competent Bacteria

In the transformation process, competent bacteria were stored at -80°C in 100 microliter aliquots. For each
transformation, 50 μl aliquots of competent cells were used. 1 μl of each plasmid (pSB1C30 and pJUMP26-1A)
was transformed.

The cells were initially incubated on ice for 30 minutes. A heat shock was then performed, involving a brief
exposure to 42°C for 40 seconds, followed by immediate placement back on ice for 2 minutes. Subsequently,
950 μl of LB medium were added to the cells. The cells were then shaken at 37°C for 1 hour. To complete the
transformation process, 250 μl of the transformation solution were plated on the plates with the
corresponding antibiotic (chloramphenicol for pSB1C30 and kanamycin for pJUMP26-1A).

## 1.6.Transformation of Plasmids and Colony Selection

We noted that the transformation of plasmids (pSB1C30 and pJUMP26-1A) resulted in colonies appearing only on
the pJUMP26-1A plates. The following day, all bacterial colonies from the transformed plates were spun down
and plated using 250 microliters of the bacterial culture. Colonies were observed on both types of plates
this time, with pSB1C30 colonies notably displaying a distinctive red coloration, which confirmed that the
transformation had succeeded, as pSB1C30 contains a red fluorescent protein. The pJUMP26-1A colonies had a
green tint, which could also be thanks to the green fluorescent protein that the plasmid contains. Three
selected colonies from both types of plates were grown overnight at 320 RPM and 37°C.

![](https://static.igem.wiki/teams/4740/wiki/zzfig3expa.jpg)

*(a)*

![](https://static.igem.wiki/teams/4740/wiki/zzfig3expb.jpg)

*(b)*

*Fig.3 Transformation of plasmid DNA for pSB1C30(a) and pJUMP26-1A(b)*

## 1.7.DNA Extraction Using QIAprep Miniprep

The DNA extraction process involved several steps to obtain purified DNA samples for further analysis.The
bacterial cells were initially spun down at 5400 g for 10 minutes at 4°C and the pellet was then resuspended
in 250 μl of Buffer P1. Subsequent steps involved the addition of Buffer P2, Buffer N3, centrifugation, and
washing to remove impurities and elute the DNA. We added 250 μl of Buffer P2 and mixed thoroughly by
inverting the tube 4–6 times. Then we added 350 μl of Buffer N3, mixing by inverting the tube 4–6 times. We
then centrifuged for 10 minutes at 13000 rpm (~17,900 x g) in a table-top microcentrifuge and applied 800 μl
of the supernatant to the QIAprep 2.0 Spin Column by pipetting. All the following centrifugations were done
at 13000 rpm. Next we centrifuged for 1 minute and discarded the flow-through. After that we washed the
QIAprep 2.0 Spin Column by adding 0.5 ml of Buffer PB and centrifuging for 1 minute, then discarding the
flow-through. We then washed the QIAprep 2.0 Spin Column by adding 0.75 ml of Buffer PE and centrifuging for
1 minute and discarded the flow-through and centrifuged for an additional 1 minute to remove residual wash
buffer. Then we placed the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, we
added 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) and water to the centre of each QIAprep 2.0 Spin Column and
let it stand for 1 minute.It was then centrifuged for 1 minute.

## 1.8.Quantification of DNA

Quantification of DNA was carried out using the Simplified QuantiFluor protocol. Samples were incubated at
room temperature for 5 minutes before readings were taken. Standard readings were recorded as 204 ng/ul. For
pSB1C30, we determined a concentration of 54 ng/ul and for pJUMP26-1A, a concentration of 16 ng/µL.

## Digestion of Plasmids

Digestion of pSB1C30 and the pJUMP26-1A was carried out using EcoRI and PstI. The expected sizes of
resulting fragments were 2029 bp for pSB1C30, and 2260 bp for pJUMP26-1A. The digestion mixture included 1
μl of DNA, 4 ul of 10x buffer, 1.5 μl of EcoRI, 1.5 μl of PstI, and 8 μl of water. Incubation occurred for 1
hour at 37°C for both plasmids.

## Agarose Gel Electrophoresis

Gel electrophoresis was run at 10 V/cm, with an agarose gel prepared using 0.5 grams of agarose in 50 ml of
TEAE 1X. Ethidium Bromide was added to the gel to visualise DNA. Electrophoresis was conducted using a
Nahira gel box at 100 V. This step confirmed that the fragments were those desired as they corresponded to
the expected sizes.

![](https://static.igem.wiki/teams/4740/wiki/zzfig4.png)

*Fig.4- Digested DNA fragments on agarose gel electrophoresis*

## Gel Loading Dye (10x)

To facilitate loading DNA samples onto the agarose gel, a 10x gel loading dye was prepared. It consisted of
1 ml of DNA-loading Buffer, 500 μl of 50% glycerol (diluted in DEPC-treated water), 20 µl of 0.5 M EDTA (pH
8.0), and 480 μl of H2O. Additionally, 2.5 milligrams of bromophenol blue were dissolved in 100 millilitres
of the solution, resulting in a concentration of 2.5 milligrams per millilitre of the dye solution.

## Cloning Protocol

As a first-time iGEM team, we experienced problems with creating a cloning protocol that would actually be
successful. Because of this delay, the DNA fragments didn’t arrive in time. We only had time to do the steps
above, up to the point of the actual insertion of the genes into the plasmids (the last step we did was
restriction digest of plasmid DNA), but we did thoroughly research the entire cloning strategy we proposed
and we hope it will help future teams.

## Cloning Strategy

**Aims**

Aim 1- Clone the transcription factor alkS under the control of a constitutive promoter

Aim 2 – Clone alkB1 under the control of alkS-inducible palkB promoter

**Genes to clone**

· alkS- Transcription factor involved in the expression of alkane-degrading genes. In the presence of
alkanes, it binds palkB and inducing start of transcription.

· palkB – alkS-inducible promoter for alkB1

· alkB1 - Catalyzes the hydroxylation of n-alkanes and fatty acids in the presence of a NADH-rubredoxin
reductase and rubredoxin.

**A. Cloning constitutive promoter + alkS**

· pJUMP26-1A (from the distribution kit plate 2 – well E17) as backbone (Kanamycin resistance)

· constitutive promoter J23119(order from IDT: prefix- J23119 -suffix)–suitable for bacterial and
cyanobacterial systems

· alkS from IDT (prefix-RBS-alkS-suffix)

**STEP 1: open pJUMP26-1A**

1. Digest backbone pJUMP26-1A with EcoRI and PstI

2. Run gel electrophoresis of digestion reactions

3. Isolate part of interest by gel digestion

**STEP 2: digest promoter J23119**

4. Digest promoter J23119 with EcoRI and PstI

5. Clean the digestion reaction by PCR clean-up

**STEP 3: digest alkS**

6. Digest alkS with XbaI and PstI

7. Clean the digestion reaction by PCR clean-up

**STEP 4: insert promoter J23119 in the open pJUMP26-1A plasmid**

8. Ligate open pJUMP26-1A and J23119 using T4 ligase

9. Bacterial transformation

10. Miniprep

**STEP 5: open pJUMP26-1A – J23119**

11. Digest pJUMP26-1A – J23119 with SpeI and PstI

12. Clean the digestion reaction by PCR clean-up

**STEP 6: insert alkS in the open pJUMP26-1A – J23119**

13. Ligate alkS into pJUMP26-1A – J23119 using T4 ligase

14. Bacterial transformation

15. Miniprep

**B. Cloning alkS-inducible promoter pAlkB + alkB1**

· Plasmid pSB1C30 (from the distribution kit plate 1 – well O11) as backbone (Chloramphenicol resistance)

· alkS-inducible promoter pAlkB from IDT (prefix-pAlkB-suffix)

· alkB1 from IDT (prefix-RBS-alkB1-suffix)

**Steps**

1. Digest pSB1C30 with EcoRI and PstI

2. Digest pAlkB (from IDT; prefix-pAlkB-suffix) with EcoRI and PstI

3. Digest alkB1 (from IDT; prefix-RBS-alkB1-suffix) with XbaI and PstI

4. Run gel electrophoresis of digestion reactions

5. Isolate parts of interest by gel digestion

6. Ligate openpSB1C30 and pAlkB using T4 ligase

7. Bacterial transformation

8. Miniprep

9. Digest pSB1C30-pAlkB with SpeI and PstI

10. Clean the digestion reaction by PCR clean-up

11. Ligate open pSB1C30-pAlkB and AlkB1 with T4 ligase

12. Bacterial transformation

13. Miniprep

**C. Cloning promoter J23119 + AlkS in plasmid with promoter pAlkB + AlkB1**

· Plasmid pJUMP26-1A with promoter J23119 +AlkS (from step A)

· Plasmid pSB1C30 with pAlkB+alkB1 (from step B)

**Steps**

1. Digest plasmid pJUMP26-1A with promoter J23119 +AlkS with XbaI and PstI

2. Digest plasmid pSB1C30 with pAlkB+alkB1 with SpeI and PstI

3. Run gel electrophoresis of digestion reactions

4. Isolate parts of interest by gel digestion

5. Ligate open pSB1C30and pAlkB+alkB1 using T4 ligase

6. Bacterial transformation

7. Miniprep and sequencing(using standard primers VF2 and VR)

## Standard protocols

**1. Digestion**

Add components to a clean tube in the order shown:

1. 1 µL DNA (concentration 1 µg/µL).

2. 2.5 µL 10x buffer CutSMART (or recommended buffer from NEB).

3. 1 µL each restriction enzyme.

4. 15 µL nuclease-free water.

5. Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.

6. Stop the digestion by heat inactivation at 65°C for 15 minutes (temperature may vary depending on which
restriction enzymes were used).

7. At this point, samples may be stored at −20°C.

Alternatively, run DNA electrophoresis and proceed with gel extraction.

**2. Ligation**

Add components to a clean tube in the order shown:

1. Add 2 µL 10x reaction buffer for T4 DNA ligase.

2. Add 1 µL of T4 DNA ligase.

3. Add 60 ng DNA in total (mix of parts to ligate).

4. Add the necessary amount of nuclease-free water to give a final volume of 20 µL.

5. Incubate at room temperature (~22°C) for 30 min.

6. Heat-inactivate the T4 ligase by heating at 80°C for 20 min.

7. At this point, samples may be stored at −20°C.

Alternatively, run DNA electrophoresis and proceed with gel extraction.

**3. DNA electrophoresis**

Gel preparation - Casting a 150 ml gel

1. Set the DNA cast to the sealed position (depends on equipment)

2. For a 1% 150 mL agarose gel, weigh 1.5 g of agarose in a 500 mL conical flask. Add 150 mL 1x TAE/TBE
buffer. If the gel tray is smaller, calculate the amount needed.

3. To dissolve the agarose in the buffer, swirl to mix and microwave for a few minutes taking care not
to
boil the solution out of the flask. Agarose has to be completely dissolve in the buffer and therefore, the
solution should be transparent. In order to do this, make the solution boil for a few seconds, swirl and
boil again. Be careful — the solution is very hot! Insulated gloves are too bulky to easily pull the flask
out of the microwave, so a folded up paper towel is suggested.

4. Let the agarose solution cool down. Once the flask is touchable, add the DNA stain. The working
concentration for Sybr®Safe is 1x (15 uL for 150mL gel).

5. Pour the gel solution into the gel tray. Remove any air bubbles with a pipette tip. Insert the comb
into
the gel tray at one end ~1 cm from the edge.

6. Let the gel solidify by cooling down to room temperature during ~30 min.

**4. Run agarose gel**

1. Release the gel tray from the casting stand. Place the gel tray into the buffer chamber and remove the
comb carefully.

2. Add 1x TAE/TBE buffer until the gel is completely covered.

3. Take part of your DNA samples (~0.2 ug) and mix with loading dye. This can be done either in 1.5 mL
tubes
or, if the volumes are very small, on a piece of parafilm.

4. Load the size marker mixed in 1x loading dye (~6 uL final volume) into the first well. If you have
many
samples, you might load size markers in additional wells.

5. Load your samples into the other wells. Remember to write down which lanes have which samples.

6. Put the lid onto the buffer chamber and connect it to the power supply. Make sure to put it in the
right
direction so that your DNA runs towards the positive (red) electrode.

7. Run the gel at 100 V for 30-60 min. Neither of the two dyes should be run off the gel. If the
electrophoresis runs correctly you will notice air bubbles coming from the negative (black) electrode.

8. Stop the run and bring your gel to a UV table to visualise your gel bands. Take a picture of your
gel. If
sufficient separation of the bands was not achieved, put the gel back into the buffer chamber and run it for
longer.

**5. Gel digestion**

1. Take your gel to the UV table to visualise your gel bands.

2. Take a scaple and cut out the bands of interest. Remove as much agarose as possible.

3. Put each band in an eppendorf tube.

4. Proceed with gel digestion protocol following kit instructions.

5. Elute with 20 uL elution buffer or nuclease-free water.

6. Measure concentration and purity of eluted DNA.

## References

[1]
 [*Josefine Liljeruhm, Erik Gullberg, Anthony C. Forster. Synthetic
biology. A lab manual*](https://zlib.pub/book/synthetic-biology-a-lab-manual-g0epq58cf0c0)

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## Results

Pioneering sustainable solutions at the forefront of synthetic biology,
revolutionizing our world one gene at a time.

[See results](#rezzz)

* Chestie 1
* Chestie 2
* Chestie 3
* Chestie 4
* Chestie 5

## Inspiration

"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.""Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

---

## Our solution

"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

### Erythritol utilizing pathway

"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

### Erythritol induction system

\"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

### Platform applications

"Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum."

{% endblock %}