iGEM 2023 — Team groningen

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}Description{% endblock %} {% block page\_content %}

## Notebook

On this page we have documented our weekly wet lab progress during our project. The purpose of this Notebook is to outline which experiments
were done when, and also to present some of the preliminary results. To see the final results in detail, please visit the
[Results Biology](/groningen/results-biology) page.

To learn more about our engineering approach, go to the
[Engineering](/groningen/engineering) page. To view our full timeline please refer to the following
[PDF](https://drive.google.com/file/d/12s6O_paqaGVXWkFaMVkgCcZfOZu9U6U_/view?usp=drive_link).

## Weekly Summaries

## August 7 - 13

* Made sure everything we need is in the lab
* Made media, autoclaved tips, and other materials to prepare for experiments

## August 14 - 20

* We completed the interlabs calibration protocol and prepared experiment 2 of interlabs
* Obtained all designed and ordered plasmids/parts

## August 21 - 27

* Finished experiment 2 of interlabs and sent the results
* Finished cloning strategy for fluorescent biosensor plasmid
* Made & tested chemically competent E. coli DH5-alpha cells
* Mini-prepped the luciferase plasmid + promoter
* PCR of YhjH front and back, AGE backbones, CUG\_Fleq, CUG\_promoter, Luciferase cassette

## August 28 - September 3

* PCR & Cloning luciferase plasmid (2x) + promoter
* Analyzed results of last week’s PCR’s on agarose gel
* First attempt of Gibson assembly of the CUG promoter in an empty plasmid
* Second attempt at previously failed Luciferase cassette PCR
* Band purification using the Thermo Scientific GeneJET PCR purification kit
* Made more competent cells
* Colony PCR CUG promoter
* Gibson assembly of the c-di-GMP hydrolase
* Received a dilution streak of a TG1 glycerol stock
* PCR\_phusion of the backbones
* Tested competency of both DH5-alpha and TG1 cells
* Digestion of backbones
* PCR cleanups
* Colony PCR

## September 4 - 10

* PCR + Cloning (Gibson Assembly) of hydrolase plasmid + promoter
* Sent promoter for sequencing
* Hydrolase plasmid: Transform into end-point cells (TG1)
* Colony PCR of promoter and hydrolase
* Send hydrolase and CUG promoters for sequencing
* Design new primers for golden gate for CUG promoter that didn’t work with Gibson Assembly
* Perform standard assembly to bind the promoter (constitutive) to the GFP and RFP Redigested GFP E+S and ligation
* Biobrick inoculation

## September 11 - 17

* Transformation of the GFP and RFP construct + colony PCR
* PCR the luciferase cassette
* Sent the RFP and GFP constructs for sequencing
* Transformed AGE bricks and UspB in E. coli DH5-alpha
* Analyze sequencing results
* Perform Golden Gate for CUG promoter
* Electroporation for dispersin B
* Mini-prepped AGE biobricks
* Restriction digest GFP

## September 18 - 24

* Mini-prepped Dispersin B transformation and sent for sequencing
* Golden gate assemblies of CUG promotor (pSB1A3\_CUG) didn’t work
* Sent results for sequencing and analyzed them
* Made new overnights and mini-prepped them
* Transformation and colony PCR CG\_CUG 1:5 and 1:10 ratios, were unsuccessful

## September 25 - October 1

* Restriction digests and ligation, GFP and RFP+C7 were successful
* Ligation of psBIC3\_GFP\_Pce+RFP transformation in E. coli DH5-a
* Discussed with supervisors why assembly of CUG promoter didn’t work. Need to redesign plasmid maps. Will try to build a construct with a
  different inducible promoter due to time constraints.
* Made electrocompetent E. coli DH5-a cells
* Isolated GFP to measure with the electronic device, the device was able to detect the isolated GFP!
* Set overnight cultures of more biobricks and mini-prepped them
* PCR of new biobricks
* Digests, ligation and transformation to obtain new constructs

## October 2 - 8

* Sent GFP+C7 samples for sequencing
* Analyzed sequencing results of GPR
* Q5 PCR BBa\_K180000+DprI and PCR clean-up
* Digested BBa\_B1006 with E+X, Digested BBa\_E0040 with E+S and ligation (pSBIC3\_GFP+Term)
* Colony PCR of ligation 02-10 (pSBIC3\_GFP+Term)
* Mini-prepped YhjH overnight cultures, performed restriction digest and analyzed results on agarose gel, obtained correct construct
* Sent GFP + Term DNA samples for sequencing
* Digestion of several constructs, ligation of correct construct fragments and transformation in E. coli DH5-a
* Fragments used for ligations:
  + 1. Ptac + BBa\_B0034 > K1800000
  + 2. Linear Ptac + pSBIC3\_GFP\_Pte+RFP > pSBIC3\_Ptac+GFP\_Pce+RFP
  + 3. CUG-FleQ + pSBIC3\_GFP+Term > pSBIC3\_CUG-FleQ\_GFP+Term
  + 4. pSBIC3\_GFP+Term + pSBIA3\_RFPdevice > pSBIA3\_GFP+Term\_RFPdevice
  + 5. FleQ + CUG + pSBIK3 > pSBIK3\_CUG
* Colony PCR for colonies of transformation with possible constructs 1, 2, 3, 4 & 5.
* Colony PCR results were ambiguous due to malfunctioning of agarose gel electrophoresis
* Made overnights of colony material or new colonies from all material containing possible constructs
* Mini-prepped overnights and performed digestion to analyze identity of constructs. Sent promising looking results for sequencing.
* Performed ligations to obtain possible new constructs and transformed them in E. coli DH5-alpha
* Fragments used for ligations:
  + A: pSBIK3\_Ptac+RBS + pSBIC3\_GFP+Term\* > pSBIC3\_Ptac+RBS+GFP+Term
  + B: Ptac (K180000) + pSB3T5 (E8) > pSB3T5\_Ptac
  + C: pSBIK3\_CUG + pSBIC3\_GFP+Term\* > pSBIC3\_CUG\_GFP+Term
* Ligations were done with multiple samples of a fragment to have a higher chance of getting the desired results.
* The c-di-GMP sensor was not obtained. Ptac+GFP+Pcl+RFP was not obtained (RFP was likely removed by recombination, because of the repeating
  sequence of terminators). Pcl+Ppel+GFP was obtained, but not pure (dilution streak with colony screening required).

[Back to Home](https://2023.igem.wiki/groningen/index.html)

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{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead %}{% endblock %} {% block page\_content %}

## Introduction - a biofilm sensor

As thoroughly discussed on the [Project Description] page, the goal of our project is to create a sensor that can measure the presence of a
biofilm on medical implants. Additionally, we also aim to include a treatment strategy that will activate upon detection of a biofilm. The
biological part of our two-component sensor is an *E.coli* biosensor containing three engineered plasmids which equip the biosensor
with the necessary characteristics to pick up the correct signal and communicate, via fluorescent light, with the second component, the
electronic sensor.

The electronic sensor can pick up the fluorescent signal and can communicate with the patient wearing the biosensor that the biofilm is
present and forming. The following sections will describe the background information and the individual design efforts performed to create
the biological component of our sensors, from now on referred to as the biosensor. An overview of all designed parts, including links to the
repository, can be found on our [Parts] page. Additionally, Figure 1 shows an overview of all constructs discussed on this page.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering01.png)

**Figure 1:** Answers regarding demographics.Figure 1: An overview of the created constructs during the Bye-o-film project, color-coded
based on the function of the construct (left) with a more detailed description of the particular constructs (right). Created using the
resources from Slidesgo and Freepik

## Introduction - saying bye to the biofilm

Our second aim of the project is to include a treatment strategy that should be activated upon detection of biofilm formation. Our treatment
is based on the Dispersin B (BBa\_K1659200) activity. Dispersin B releases cells from the biofilm via degradation of the polysaccharides,
targeting β-1,6-glycosidic linkages in poly-N-acetylglucosamine (PGA) [1][2]. This is found in the biofilm of three major
biofilm-forming bacteria E. coli, S. aureus, and S. epidermidis [1].

To deliver Dispersin B, we created a phagemid design by displaying it on the PIII protein of an M13 phage [3]. The exact design
of the phagemid will be explained in Construct 2. The activity of Dispersin B was purposefully selected using the general infection and
lysis function of the M13. This is because the M13 phage is specific to *E.coli*, but to be broadly applicable, Bye-O-Film needs to
target other biofilm-forming bacteria as well. Using the other well-studied capacity of M13 phage-display we can display Dispersin B,
utilizing its ability to degrade biofilm of not just *E.coli*, but also S.aureus and S.epidermidis.

Besides its broad applicability, the mode of Dispersin B operation includes another benefit. With the cleavage of β-1,6-glycosidic linkages,
the bacteria in the biofilm are degraded to a planktonic form. Aiding the body to naturally get rid of the infection. In the case of
antibiotic treatment, it also makes the bacteria more susceptible to antibiotics and greatly enhances the likelihood of successful
treatment. Ultimately also leading to a reduction of the required antibiotics for sufficient treatment.

## Team: CUG-China 2022 - our basis

In 2022, the CUG China iGEM team embarked on a journey to create a cyclic-di-GMP-based biosensor that can detect biofilm formation.
Bacteria, when moving from a planktonic state to form a biofilm, perform this transformation step by an increase in the release of various
small signaling molecules (quorum sensing). Among these signaling molecules is cyclic-di-GMP. CUG China envisioned using this quorum sensing
to create a biosensor that can ‘talk along’ with the biofilm formation.

CUG China ultimately used the Ppel promoter (BBa\_K4242001), which is repressed by the protein FleQ. When cyclic-di-GMP is present, it will
bind FleQ (BBa\_K4242000) and will change the effector function of FleQ from a repressor to an activator. When cyclic-di-GMP is present,
genes downstream of the PpeI promoter will be transcribed, while the absence of cyclic-di-GMP FleQ will repress the transcription of
downstream products (Figure 2).

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering02.png)

**Figure 2:** Diagram showing how the CUG-China 2022 team’s ci-di-GMPT-induced promoter works. Created with BioRender.com

The PpeI promoter is T7 dependent. CUG China used a BL21 E. coli strain with this gene. Even though their strains should be able to utilize
the PpeI promoter, they have chosen to add the constitutive PcI promoter (BBa\_R0051). In our project, we use DH5-alpha for cloning, and TG1
as our final species, due to its well-described phage display possibility. We bypassed the problem of lacking the T7 gene by using the same
constitutive PcI promoter. In Construct 1, we will describe how the presence of this constitutive promoter allows us to perform a
fundamental check on the activity of our biosensor.

## Construct 1: Reporter module (GFP + RFP)

With the CUG sensor as a basis, different reporter options were considered. We were informed about various options through our Human
Practices endeavors. We looked into ideas such as measuring the redox potential of hydrogen peroxide by catalase activity, using the
expression of streptavidin, and measuring the output via surface plasmon resonance. Lastly, we were also advised on bioluminescence and
fluorescence as reporter signals.

The catalase idea was soon disregarded as impractical, because of complications of measuring the redox potential in the complex environment
of the body. The Surface Plasmon Resonance technique was abandoned due to price considerations. The other options, using luciferase and
fluorescence, were especially attractive taking the recommendations as mentioned in the [AREA Framework] into account. Our efforts to use a
luciferase cassette from plasmid pAKgfplux1 (Addgene Plasmid #14083) were postponed. The size of the cassette proved difficult during our
cloning strategy. The applied strategy is further denoted in Construct 1C.

Ultimately, the choice was made to utilize GFP (BBa\_E0040). Not only is GFP much smaller, but it is also well characterized, has a stronger
signal compared to luciferase, and is readily available. This design does create extra challenges for the electrical component. How will the
light be excited? How will the sensor only pick up the correct emission wavelengths of GFP and RFP? These questions are answered on our
[Electronics page].

Our next design consideration is, then, how to normalize the expression of GFP to a useful signal. Normally GFP emission is normalized by
cell density (OD600). In our setup, where our biosensor should give output to an electronic sensor inside the body, it is not possible to
apply this strategy. Therefore, we decided to express RFP (BBa\_E1010) behind a constitutive PcI promoter (BBa\_R0051). This not only allows
us to normalize the GFP expression by the RFP expression, but it also allows us to assess the biosensor's viability by verifying the initial
presence of RFP expression. To test the hypothesis that RFP could be used to normalize GFP expression, the inducible promoter Ptac
(BBa\_K180000) was put in front of the GFP. This construct was however not obtained due to recombination around the repeating terminator
sequence, which resulted in the removal of the Pcl-induced RFP from the construct.

Another design consideration we tackled was the issue of varying promoter strengths between the constitutive promoter PcI and the FleQ
repressed PpeI promoter. To bypass this problem, and to ensure both GFP and RFP are transcribed with the same promoter strength, the
aforementioned PcI promoter was placed in front of the PcI controlled GFP. Putting both RFP and GFP under control of the constitutive PcI
promoter, with the notable difference that GFP is also repressed by the cyclic-di-GMP dependent repression of FleQ, via the PpeI promoter
region.

Taking these considerations into account, we initially came to the following part (Figure 3):

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering03.png)

**Figure 3:** Plasmid map of initial Construct 1 (Part code: BBa\_K4720015, Part composition: BBa\_J34801 (RBS) + BBa\_E0040 (GFP) +
BBa\_B0015 (Terminator sequence) + BBa\_R0051 (PcI promoter) + BBa\_B0034 (RBS) + BBa\_E1010 (RFP) + BBa\_B0015 (Terminator sequence)).
[Benchling link](https://benchling.com/s/seq-vUl0WTTyzMF1Fc68prlY?m=slm-9vmuArjxzrkf3pPx3BOq)

The individual parts were obtained from previous iGEM distributions and the constructs were obtained by bio brick RFC[10] standard
assembly.  This design incorporates RFP downstream of the constitutive promoter PcI, along with a Ribosome Binding Site (RBS). GFP, on the
other hand, is positioned downstream of a distinct RBS. It is important to note that the design currently lacks the constitutive PcI and
repressible PpeI promoters. Before introducing the PcI and PpeI promoters, two other improvements were desired.

Envisioned improvements were the replacement of one of the double terminator (BBa\_B0015) sequences with another terminator (BBa\_B1006). By
removing this sequence repetition, the construct should be less prone to recombination. Due to the time-consuming process of cloning and the
need to switch cloning strategies, we were only able to create the following part, excluding the RFP expression system.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering04.png)

**Figure 4:** Plasmid map of final progress made on Construct 1 (Part code: BBa\_K472001, Part composition: BBa\_E0040 (GFP) + BBa\_B1006
(Terminator sequence)). [Benchling link](https://benchling.com/s/seq-3zJEPHeLjXJAznMoeHUF?m=slm-GmPFiJCPhLybUA9FfEQw)

When part BBa\_K4720012 gets incorporated into the original BBa\_K4720015, the envisioned theoretical part would be created:

| Part Code | Part Composition | Benchling Link |
| --- | --- | --- |
| BBa\_K4720023 | BBa\_E0040 (GFP) + BBa\_B1006 (Terminator) + BBa\_R0051 (PcI promoter) + BBa\_B0034 (RBS) + BBa\_E1010 (RFP) + BBa\_B0015 (Terminator sequence) | [Benchling Link](https://benchling.com/s/seq-R6xGxs7C0Mq0OSqAZDLb?m=slm-x7JVZQBCPYHZmySLH4MD) |

#### Construct 1B(1-3) Reporter control

Our final sensor product, Construct 4, ultimately uses the reporter function as described in Construct 1 and the sensor function as
described in Construct 3. As earlier described, the expression of GFP will be under cyclic-di-GMP dependent FleQ control. A potential
prospect of this final construct is that GFP will never be expressed. To check if it is possible to express GFP in our design, and to
potentially pinpoint future problems to the repression and/or activation of FleQ, we created Construct 1B1.

Construct 1B1 (BBa\_K4720019), therefore resembles the promoter-deficient GFP intermediate from Construct 1. Construct 1B1, in
comparison, includes the IPTG inducible promoter Ptac (BBa\_K180000). Firstly, with this construct, we can test if we can produce GFP in this
system, without the PpeI promoter. Secondly, we can use this construct to measure the potential presence of Fluorescence Resonance Energy
Transfer (FRET) effects. When taking the excitation and emission spectra into account for both GFP (BBa\_E0040) and RFP (BBa\_E1010), it is
possible to see quenching of GFP in the presence of RFP. The opposite, quenching of RFP due to the presence of GFP should not be measured.

To measure this we created, besides Construct 1B1, two other constructs. Construct 1B2 contains an RFP behind a
constitutive PcI promoter. Construct 1B3, our dual GFP and RFP expression system contains both fluorescent proteins. In principle, the
expression of RFP, under the same conditions, should be the same between Constructs 1B2 and 1B3. As well as the expression of GFP when the
same amount of IPTG is added to induce Ptac in 1B1 and 1B2. The difference in measured fluorescence between 1B3 - 1B1 shows the level of
quenching of GFP by RFP, while the measured difference between 1B3 - 1B2 shows the level of quenching of RFP by GFP.

| Part Code | Part Composition | Benchling Link |
| --- | --- | --- |
| BBa\_K4720013 | BBa\_J34801 (RBS) + BBa\_E0040 (GFP) + BBa\_B0015 (Terminator sequence) | [Benchling Link](https://benchling.com/s/seq-7rGcf5J0QUhY0qPmpdZc?m=slm-KG8wgkzB2y4tURklfAaR) |
| BBa\_K4720024 | BBa\_R0051 (PcI promoter) + BBa\_B0034 (RBS) + BBa\_E1010 (RFP) + BBa\_B0015 (Terminator sequence) | [Benchling Link](https://benchling.com/s/seq-p3SDR4QNA0SSA0NqwhrB?m=slm-gUl8KAphrIUbGj6Y02MQ) |
| BBa\_K4720016 | BBa\_K180000 (Ptac) + BBa\_J34801 (RBS) + BBa\_E0040 (GFP) + BBa\_B0015 (Terminator sequence) + BBa\_R0051 (PcI promoter) + BBa\_B0034 (RBS) + BBa\_E1010 (RFP) + BBa\_B0015 (Terminator sequence) | [Benchling Link](https://benchling.com/s/seq-L9OwlPerpCda2TKrEY3j?m=slm-XF7mLABCbAS7PO55IN7M) |
| BBa\_K4720019 | BBa\_K180000 (Ptac) + BBa\_B0034 (RBS) + BBa\_E0040 (GFP) + BBa\_B1006 (Terminator sequence) | [Benchling Link](https://benchling.com/s/seq-q7A2do9MiHBGir14HjWD?m=slm-NTYpXx9EVRyQrI4CESZ2) |

#### Construct 1C Reporter module (Luciferase - Theoretical)

The plasmid containing the luciferase cassette was obtained from addgene (pAKgfplux1). Because the cassette contains an XbaI restriction
site, site-directed mutagenesis is required to make the part RFC[10] compatible. The primers, Figure 5 and Table 1, were designed with a 38
bp overlapping region for circularization, and were designed to amplify the new cassette for insertion into the pSB1A3 vector by Gibson
Assembly.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering05.png)

**Figure 5:** Primers designed for site-directed mutagenesis to remove XbaI restriction site making the construct RFC[10] compatible.

| pSB1A3\_Backbone\_Fwd | TACTAGTAGCGGCCGCTGCAG |
| --- | --- |
| pSB1A3\_Backbone\_Rev | TCTAGAAGCGGCCGCGAATTCC |
| Lux\_Fwd | cttgcccttttttgccggactgcagcggccgctactagtaTCAACTATCAAACGCTTCGGTTAAGC |
| Lux\_Rev | ttcgctaaggatgatttctggaattcgcggccgcttctagATGACTAAAAAAATTTCATTCATTATTAACGGCCAGG |
| MutLux\_Fwd | CATTAATGAATTGCCGAATAACCTAGATTTTGAAGGCCATAAATTGGGTGCTGAAGTC |
| MutLux\_Rev | GGCCTTCAAAATCTAGGTTATTCGGCA ATTCATTAATGGGTAGACTGAGATAATCAAACCC |

**Table 1:** Overview of primer pairs designed for site-directed mutagenesis (MutLux), primer pair for amplification of the Luciferase
cassette (Lux), and primer pair for amplification of the pSB1A3 Backbone: pSB1A3\_Backbone.

After the Gibson assembly of this theoretical module, we could have combined Construct 1C (reporter module) with Construct 3 (sensor
module), to generate the following part:

| Part Code | Part Composition | Benchling Link |
| --- | --- | --- |
| BBa\_K472010 | J23100 + RBS + FleQ + TT + Pcl + Ppel + RBS + Lux | [Benchling Link](https://benchling.com/s/seq-u1crDaynJrWE1On550OK?m=slm-BrGE0MyOHYfhzJiXbNJF) |

## Construct 2: Hydrolase

*E.coli* naturally produces cyclic-di-GMP: which means a biosensor for this molecule cannot be tested, as the promoter will always be
active. To circumvent this, we used the same approach as the CUG\_China 2022 team: overexpression of the cyclic-di-GMP hydrolase to reduce
the endogenous cyclic-di-GMP concentrations.

We also explored the option of knocking out genes responsible for cyclic-di-GMP production in our cells, but we soon realized that the
expression system for this signaling molecule is more complex and cannot be easily suppressed [4].

To reduce the intracellular cyclic-di-GMP concentration, we used the cyclic-di-GMP hydrolase yhjH (BBa\_K861090) with the IPTG inducible
promoter Ptac (BBa\_K180000) used by the CUG\_China 2022 team in the brick: BBa\_K4242017. No alterations were made to this brick. The
construct was placed in the pSB3T5 vector. This backbone was chosen because of the compatibility of the origins of replication and the
difference in antibiotic resistance marker compared to the biosensor construct containing vector pSB1C3.

The insert was obtained as two synthetic DNA fragments from TWIST bioscience. The construct was successfully obtained by Gibson Assembly
into the pSB3T5 vector.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering07.png)

**Figure 6:** Plasmid map of Construct 2 (Part code: BBa\_K4242017). Part composition: BBa\_K180000 (Ptac) + BBa\_J3480 (RBS) +
BBa\_K861090 (YhjH) + BBa\_B0015 (TT)[Benchling link](https://benchling.com/s/seq-CHG1NBC0FNbPpp6tXVdZ?m=slm-IN8L7HjJWf56sNwBK6y1)

#### Construct 2B Hydrolase control

With the introduction of Construct 2, we are not only introducing the cyclic-di-GMP hydrolase but also the IPTG inducible Ptac. To control
the hydrolase activity of Construct 2, Construct 2B was created. The control contains the Ptac promoter (BBa\_K180000) but does not contain
the hydrolase. Again the pSB3T5 was used as a backbone.

| Part Code | Part Composition | Sequence Link |
| --- | --- | --- |
| BBa\_K4720022 | BBa\_K180000 (Ptac) + BBa\_J3480 (RBS) + BBa\_B0015 (Terminator) | [Benchling Link](https://benchling.com/s/seq-NEVM9uFTFXHqJkWxZaH7?m=slm-oDdiENbQCbg3XBlLG9c3) |

## Construct 3: Sensor module (PpeI + FleQ)

The original design of the cyclic-di-GMP sensor was obtained from the 2022 CUG China team (BBa\_K4242013). This design was ordered as
synthetic DNA from TWIST bioscience in two parts. A codon optimization was performed on the FleQ coding sequence (BBa\_K4720000). During the
optimization, three SapI restriction sites were removed, making the part RFC[1000] compatible. The fragments were designed with 30 bp
overlap at the ends for Gibson Assembly into the pSB1A3 vector, the final product is shown in Figure 7. The colonies transformed with the
Gibson Assembly reaction mix were screened by colony PCR, without any result. In the samples, the FleQ coding sequence was consistently
absent when reviewing the sequencing results.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering08.png)

**Figure 7:** Plasmid map representation of Construct 3, the sensor module, designed for Gibson Assembly. Green: Pcl+RBS+FleQ. Blue:
Double terminator+Pcl+Ppel+RBS. Red: pSB1A3 backbone. Part code: BBa\_K4720021, part composition: BBa\_J23100 + BBa\_BBa\_K4242000 (FleQ)
BBa\_B0015 (TT) + BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) + BBa\_J34801 (RBS)

A second attempt to obtain the cyclic-di-GMP sensor was made with SapI Golden Gate Assembly using the same codon-optimized fragment. The
previously mentioned synthetic DNA fragments were amplified by Q5 PCR and the SapI recognition sites were added with the primer overhangs.
The fragment sizes were confirmed on a 0.9% agarose gel. The colonies obtained from the transformation with the Golden Gate reaction mix
were screened by colony PCR and restriction digests. None of the colonies contained the desired construct. After sequencing it was concluded
that some samples contained empty backbones, while others contained only the promoter insert. The possibility of just the insertion of the
promoter in the ligation with pSB1A3 is significant. Only one of the bases of the sticky ends is not matching (Figure 8). An explanation
could be that FleQ circularizes because there is one mismatching base. After consultation with our supervisor, Julia, it was concluded that
a possible explanation could be that the concentration of FleQ is lowered due to the circularization. To test this hypothesis a second
attempt with the SapI Golden Gate strategy was carried out, but instead of equal molar ratios, the insert-to-backbone ratio was now tried at
both 5:1 and 10:1. As the second attempt was unsuccessful, we sought the help of another supervisor, Patricia. During this meeting, it was
hypothesized that the repeating sequence of the PcI promoter (which is both in front of the FleQ and the PpeI promoter) caused
recombination, resulting in the deletion of FleQ.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering09.png)

**Figure 8:** The three fragments with sticky ends were used in Golden Gate assembly with SapI. Created with BioRender.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering10.png)

**Figure 9:** Ligation of pSB1A3 with only Promoter as an insert. Created with BioRender.

| FleQ\_SapI\_Fwd | gatgctCTTCTAGAGTAACACCGTGCGTG |
| --- | --- |
| FleQ\_SapI\_Rev | gatgctctTCGTCACGCCGACTCATACC |
| Promoter\_SapI\_Fwd | gttgctcttCGTGACGACGACCTGTCGGATG |
| Promoter\_SapI\_Rev | cgggctcttcTAGTATTTCTCCTCTTTCTCTAGTACGGAAGCATGG |

**Table 2:** Overview of primers used to amplify the fragments used for the SapI Golden Gate Assembly. Lowercase bases are primer
overhangs.

A third design was made for a Golden Gate Assembly with BsaI. BsaI cleavage results in sticky ends of 4 bases, instead of the 3 bases with
SapI. This increases the amount of possible combinations from 64 to 256. Because the pSB1A3 vector contains a BsaI restriction site in the
ampicillin resistance marker, the construct was designed with the pSB1K3 vector. In order to remove the repeating sequence, the Pcl promoter
in front of FleQ was replaced by the high-strength BBa\_J23100 from the Anderson constitutive promoter family. As a high concentration of
FleQ is required to detect above-native levels of cyclic-di-GMP, a strong promoter is required. Additionally, the BBa\_J23100 has been well
characterized in E. coli TG1 (http://parts.igem.org/Part:BBa\_J23100). This new promoter sequence was placed on the primer overhangs and the
fragment was amplified behind the Pcl promoter in order to remove it. All the fragments required for the assembly were successfully obtained
by Q5 PCR, as confirmed by 0.9% agarose gel. Colonies obtained after transformation with the assembly reaction mix were screened by colony
PCR and restriction digest (NotI) but no right fragment sizes were identified.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering12.png)

**Figure 10:** Binding site of FleQ\_Pro\_BsaI\_Fwd primer on synthetic DNA fragment. Image from Benchling.

| Promoter\_BsaI\_Fwd | gatggtctcCGGCGTGACGACGACCTGTCGGATG |
| --- | --- |
| Promoter\_BsaI\_Rev | gatggtctcTGCAGCGGCCGCTACTAGTATTTCTCCTCTTTCTC |
| FleQ\_Pro\_BsaI\_Fwd | gatggtctcacctaggtacagtgctaGCTACTAGAGAAAGAGGAGAAATACTAGATGTGGCG |
| FleQ\_BsaI\_Rev | gaaggtcTCACGCCGACTCATACCATATTTACGCATC |
| pSB1K3\_BsaI\_Fwd | ctaggtctCGCTGCAGTCCGGCAAAAAAGGGCAAGGTG |
| pSB1K3\_Pro\_BsaI\_Rev | gttggtctcataggactgagctagccgtcaaCTCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCG |

**Table 3:** Primers used to amplify the fragments for Golden Gate Assembly with BsaI.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering14.png)

**Figure 11:** The sticky ends of the fragments created by BsaI digest for Golden Gate Assembly. Additional effort was put in to reduce
the compatibility of the sticky ends between undesired combinations.

## Construct 3B: Sensor control module (PpeI + K.O. FleQ)

In Construct 1B, we aimed to assess our capability to express both GFP and RFP. Additionally, we sought to create a control scenario in
which our sensor module could be tested without FleQ repression. Although a cloning strategy was created for the creation of Construct 3B,
we accidentally obtained this part (BBa\_K4720018) via recombination as described in Construct 3. The PcI repetition in front of both the
FleQ and the PpeI promoter resulted in recombination and the subsequent deletion of FleQ. This event further demonstrated the importance of
replacing one of the terminator sequences earlier described in Construct 1.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering15.png)

**Figure 12:** Plasmid map of Construct 3B (Part code: BBa\_K4720018). Part composition: BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) +
BBa\_J34801 (RBS) [Benchling link](https://benchling.com/s/seq-LGsmf0dL2mfHt2m807lm?m=slm-dNOidPbkaa4W417H8v5d)

| Part Code | Part Composition | Sequence Link |
| --- | --- | --- |
| BBa\_K4720018 | BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) + BBa\_J34801 (RBS) | [Benchling Link](https://benchling.com/s/seq-LGsmf0dL2mfHt2m807lm?m=slm-dNOidPbkaa4W417H8v5d) |

## Construct 4: Biosensor module (Construct 1+3)

To create Construct 4, our finalized biosensor module, our reporter module (described at Construct 1) and our sensor module (described at
Construct 3) needed to be combined. As described, we were unable to obtain Construct 3 due to recombination problems resulting from the use
of various repeating sequences. If we had been successful we would have been able to put a truncated version of Construct 3 (lacking the RFP
element) before Construct 1 (GFP + T) and subsequently test the construct. The theoretical part is denoted in the part registry as
BBa\_K4730021.

![](https://static.igem.wiki/teams/4720/wiki/groningen/engineering-page-img/engineering16.png)

**Figure 13:** Plasmid map of designed Construct 4 (Part Code: BBa\_K4720021). Part composition: BBa\_J23100 + BBa\_K4720000 (FleQ)
BBa\_B0015 (TT) + BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) + BBa\_J34801 (RBS) + BBa\_E0040 (GFP) + BBa\_B1006 (Terminator).
[Benchling link](https://benchling.com/s/seq-TVOgkfLvhWfT3NRtl4b2?m=slm-62Il77OqUVtfnQS0ppil)

| Part Code | Part Composition | Sequence Link |
| --- | --- | --- |
| BBa\_K4720021 | BBa\_J23100 + BBa\_K4720000 (FleQ) BBa\_B0015 (TT) + BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) + BBa\_J34801 (RBS) + BBa\_E0040 (GFP) + BBa\_B1006 (Terminator) | [Benchling Link](https://benchling.com/s/seq-TVOgkfLvhWfT3NRtl4b2?m=slm-62Il77OqUVtfnQS0ppil) |

Ultimately, our biosensor should contain the full length of our sensor and reporter modules. Above, our final product is described. However,
the RFP under the control of a constitutive promoter is still missing. Additionally, in the current design of our final construct, there is
still the risk of recombination, as seen throughout various of our experiments. Currently, FleQ, the GFP and the RFP fragments are all
under, partial, control of the constitutive promoter PcI. Besides this, there is also the issue of the double use of terminator BBa\_B0015.

As an alternative to the terminator BBa\_B0015, terminator BBa\_1002 has been selected. To replace the constitutive promoters, both BBa\_J23111
and BBa\_J23118 have been identified. These promoters are of interest due to their similarities in promoter strength (see Anderson
collection). Note that there is homology between the three Anderson promoters used, but the promoters are shorter than the PcI promoter and
there are small variations. This will need to be confirmed with additional experiments, but the thought is that these changes are sufficient
to overcome the problem of recombination.

| Part Code | Part Composition | Sequence Link |
| --- | --- | --- |
| BBa\_K4720021 | BBa\_J23100 + BBa\_K4720000 (FleQ) BBa\_B0015 (TT) + BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) + BBa\_J34801 (RBS) + BBa\_E0040 (GFP) + BBa\_B1006 (Terminator) | [Benchling Link](https://benchling.com/s/seq-TVOgkfLvhWfT3NRtl4b2?m=slm-62Il77OqUVtfnQS0ppil) |

#### Construct 4B: Biosensor control modules (Construct 1+3B, control for FleQ)

In order to test if the Pcl + Ppel tandem promoter would work in our E. coli strain TG1 we constructed the same plasmid as above, but
without the FleQ gene. This plasmid was made by the standard assembly (RFC[10]) of BBa\_K4720018 in front of pSB1C3\_BBa\_K4720012. This
construct was successfully obtained but needs purification. The sequencing results show two different sequences present behind the Pcl +
Ppel promoter, with one being right. In order to isolate the right construct, a dilution streak could be made with subsequent screening of
the isolated colonies for the right construct. This was however not done due to the time constraints.

| Part Code | Part Composition | Bencling Link |
| --- | --- | --- |
| BBa\_K4720021 | BBa\_J23100 + BBa\_K4720000 (FleQ) + BBa\_B0015 (Terminator) + BBa\_R0051 (Pcl) + BBa\_K4242001 (Ppel) + BBa\_J34801 (RBS) + BBa\_E0040 (GFP) + BBa\_B1006 (Terminator) | [Benchling Link](https://benchling.com/s/seq-IrNi1vxgrOJC7TlqHXzs?m=slm-zeKY95QHJqLdrHBvn9J5) |

## Construct 5: Phagemid (M13) + Helper Phage (Containing PIII+Dispersin B)

Our construct, a combination of Phagemid (M13) and Helper Phage (containing PIII+Dispersin B), stems from a carefully considered design,
utilizing materials from New England Biolabs and the Mayer lab from Biomolecular Chemistry & Catalysis — Stratingh Institute of Chemistry,
at the University of Groningen. New England Biolabs provided us with M13mp18 [5] , a circular double-stranded DNA derived from
bacteriophage M13, equipped with specific restriction sites within the β-Galactosidase gene. When DNA fragments are inserted into these
sites, the β-Galactosidase gene is inactivated, facilitating clone selection. Additionally, M13KO7 [6], another M13 derivative,
was used for replication in the absence of phagemid DNA. In the presence of a phagemid bearing a wild-type M13 or f1 origin, single-stranded
phagemid is preferentially packaged and secreted into the culture medium, enabling easy production of single-stranded phagemid DNA for
mutagenesis or sequencing. From the Mayer lab, we obtained phage vectors fdg3p0ss21 and fd0D1D2', provided by EPFL Lausanne.

We decided on using phage M13 as a homing device for our biofilm-degrading enzyme Dispersin B. Our design journey began with literature
research on ways to tackle biofilms, and we have stumbled upon literature indicating that bacteriophages can play this role. From that
point, we wanted to review ways in which we can utilize the phage in our project. We discussed our idea with experts, as detailed on the
[AREA Framework] and [Human Practices] pages. Our final ideas entailed M13 as a non-lytic, non-toxic human phage for delivering toxin genes,
breaking biofilms, or amplifying the light signal to our sensor, depending on which role would the phage play in the biosensor design. After
trying out different theoretical designs, our primary goal became to employ the M13 phage as a carrier for our biofilm-degrading enzyme,
Dispersin B. This choice was underpinned by M13's extensive scientific study, making it a well-explored option for creating phage display
libraries and serving as a foundational element in various applications, such as isolation, separation, sensing, probing, and in vivo
imaging [7].

M13, a filamentous bacteriophage with a single-stranded circular DNA genome, approximately 6400 base pairs long, is a versatile choice for
protein display methods. Its genome comprises ten genes, including Gene VIII, the major structural protein, and Gene III, the minor coat
protein responsible for binding to E. coli's "sex" pilus. Infection begins with the introduction of the single-stranded genome into the host
cell. Gene VIII is removed, while Gene III remains attached. Transcription and replication convert the genome into a double-stranded
circular DNA (RF form), aided by Gene II and DNA polymerase I, leading to a linear '+' genome that circularizes. Gene V prevents single
strands from reverting to RF form. During phage packaging, Gene VIII covers the M13 (+) genome [8].

M13's appeal as a cloning vector lies in its incorporation of elements like lac repressor (lac I), allowing lac promoter regulation, and a
polylinker region for fragment insertion. Sequencing and mutagenesis are facilitated by its single-stranded DNA forms, making it a valuable
tool in genetic research. This adaptability and well-studied nature make M13 an excellent choice for protein display methods, enabling the
presentation of proteins on its surface for various applications in biotechnology and research [8].

Our choice of M13 as a non-lytic phage for delivering Dispersin B aligns with our objective to target biofilm infections effectively. M13's
ability to travel to infection sites, deposit Dispersin B, degrade biofilm polysaccharides (specifically, poly-N-acetylglucosamine or PNAG),
and infect pathogenic E. coli cells makes it a preferable option to antibiotics. These infected E. coli cells can then produce more
M13+Dispersin B cells, perpetuating the cycle. To generate a full phage and control its replication, we employ a phagemid containing the DNA
coding for Dispersin B protein flanked to pIII phage protein, along with a cyclic di-GMP promoter for expression control.

We have worked towards cloning and testing this construct. However, due to logistics and temporal constraints, we were not able to obtain
conclusive results that entail our design appropriately. Instead, we provide information about the usage of M13 phagemid and helper phage
constructs, in hopes that we can enrich the ideas for future iGEM teams and bring the phage display method to light as a very versatile and
useful approach.

## In conclusion - the final plasmids

Ultimately our project would have resulted in the creation of three distinct plasmids:

1. The first plasmid would have been our combined sensor and reporter modules, a cyclic-di-GMP dependent GFP with a constitutively expressed
   RFP. This plasmid would, besides continuously expressing RFP, express GFP in the presence of the biofilm-related signaling molecule. If
   cyclic-di-GMP is sensed, it will bind to the repressor FleQ, changing its affinity for the Ppel binding site and activating the promoter
   by no longer blocking RNA polymerase from binding (BBa\_K4720106).
2. In our biofilm sensor, our E. coli biosensor will be immobilized and, thus, might be producing cyclic-di-GMP itself. Additionally, there
   is a native concentration of cyclic-di-GMP present. A problem was also noted, and tackled, by the CUG China team in 2022. Our second
   plasmid, just like CUG’s design, contains a cyclic-di-GMP hydrolase to reduce the natively present cyclic-di-GMP to ensure that our
   biosensor only activates upon binding of cyclic-di-GMP that originates from the biofilm (BBa\_K4720022).
3. Our third and final plasmid contains our treatment or response module. After many considerations on how to use the M13 phage, we settled
   on the PIII phage display of the biofilm-degrading enzyme Dispersin B. The PIII and Dispersin B are under the same cyclic-di-GMP dependent
   control as the GFP. Thus, if a biofilm is present, not only the reporter will be activated, but also our Dispersin B will be expressed. To
   be able to generate a functional M13 phage displaying the Dispersin B we will also need to infect our biosensor with a helper phage,
   containing all genes necessary to generate a fully functional M13 phage.

## Next steps

We envisioned ending the scope of our iGEM project by generating the aforementioned plasmids. However, using various plasmids in bacteria to
maintain the encoded functionality over multiple generations, poses a risk to its continuity. Over time our biosensor may reject our
plasmids, or a loss of the full set of plasmids would occur with division cycles. Therefore, the next step would be to incorporate our final
product into the genome of our biosensor, with for example CRISPR. This eventually also aids the biocontainment of our product, as there
would be no risk of our plasmids being taken up by other cells than our biosensor.

We additionally purposefully used the name ‘module’ for our sensor, reporter, and effector/treatment constructs. In principle, it is
possible to remove and replace these modules to change the reporter, sensor, or effector function of our biosensor. One additional
theoretical module, our Luciferase reporter, is one example of the possibilities of the modularity of our design.

## References

1. [Kong, W. C. (2015, August 28). Part: BBa\_K1659210. Registry of Standard Biological Parts.](https://parts.igem.org/Part%3ABBa_K1659210)
2. [Breslawec, A. P., Wang, S., Monahan, K. N., Barry, L. L., & Poulin, M. B. (2023). The endoglycosidase activity of Dispersin B is
   mediated through electrostatic interactions with cationic poly-β-(1→6)-N-acetylglucosamine. The FEBS journal, 290(4), 1049–1059.](https://doi.org/10.1111/febs.16624%C2%A0)
3. [Wang, R., Li, H. D., Cao, Y., Wang, Z. Y., Yang, T., & Wang, J. H. (2023). M13 phage: a versatile building block for a highly specific
   analysis platform. Analytical and bioanalytical chemistry, 415(18), 3927–3944.](https://doi.org/10.1007/s00216-023-04606-w)
4. [Reinders, A., Sellner, B., Fadel, F., van Berkum, M., Kaczmarczyk, A., Ozaki, S., Rueher, J., Manfredi, P., Sangermani, M., Harms, A.,
   Perez, C., Schirmer, T., & Jenal, U. (2021). Digital control of c-di-GMP in E. coli balances population-wide developmental transitions
   and phage sensitivity [Preprint]. Microbiology.](https://doi.org/10.1101/2021.10.01.462762)
5. [New England Biolabs. (n.d.). M13MP18 RF i DNA | NEB.](https://www.neb.com/en/products/n4018-m13mp18-rf-i-dna#Product%20Information)
6. [New England Biolabs. (n.d.-a). M13KO7 Helper Phage | NEB.](https://www.neb.com/en/products/n0315-m13ko7-helper-phage#Product%20Information)
7. [Wang, R., Li, H., Cao, Y., Wang, Z., Yang, T., & Wang, J. (2023b). M13 phage: a versatile building block for a highly specific analysis
   platform. Analytical and Bioanalytical Chemistry, 415(18), 3927–3944.](https://doi.org/10.1007/s00216-023-04606-w)
8. [Libretexts. (2021). 4.2: M13 Phage. Biology LibreTexts.](https://bio.libretexts.org/Bookshelves/Biochemistry/Supplemental_Modules_%28Biochemistry%29/4._Biotechnology_2/4.2%3A_M13_Phage)

[Back to Home](https://2023.igem.wiki/groningen/index.html)

{% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block lead %}Description{% endblock %} {% block page\_content %}

## Experiments

## Introduction

This page contains a collection of wet lab protocols that our team used throughout the project. The purpose of this page is to present all
the steps and reagents needed in the protocols in detail. To see the final results in detail, please go to the Results page. To learn more
about our engineering approach, head over to the Engineering page.

---

## Table of Contents

* [Growth Media](#growth-media)
* [Chemically Competent Cells (E. coli DH5-α + TG1)](#chemically-competent-cells)
* [Electrocompetent Cells (E. coli DH5-α) and Electroporation](#electrocompetent-cells)
* [E. coli Transformation](#ecoli-transformation)
* [Golden Gate Assembly Cloning using SapI](#golden-gate-assembly)
* [Phusion Polymerase Chain Reaction (PCR)](#phusion-pcr)
* [Q5 Polymerase Chain Reaction (PCR)](#q5-pcr)
* [Colony PCR](#colony-pcr)
* [Restriction/Diagnostic Digest](#restriction-digest)
* [Ligation](#ligation)
* [Agarose Gel Electrophoresis](#agarose-gel-electrophoresis)
* [Sequencing](#sequencing)
* [Antibiotics Concentrations](#antibiotics-concentrations)
* [GFP Isolation](#gfp-isolation)

---

## Growth Media

In this protocol, you can find how to make LB-Miller medium and SOB/SOC medium. SOC medium can maximize the efficiency of competent cells
during a transformation.

#### LB-Miller

*Materials:*

* LB-Miller powder.
* Demineralised water.

*Protocol:*

1. Suspend 25 g of LB-Miller powder in 1 L demineralised water.
2. Autoclave.

#### SOB/SOC

*Materials:*

* Tryptone.
* Yeast extract.
* NaCl.
* 1M KClact.
* Demineralized water.
* 1M MgCl2 solution.
* 1M MgSO4 solution.
* 20% glucose solution.

*Protocol:*

1. For 1000 mL final solution, mix the components listed in the table below.
2. Adjust to pH 7.0.
3. Fill up to 980 mL (to be completely precise), or to 1000 mL (if the previous option is not feasible).
4. Sterilize by autoclaving.
5. Before use (and after sterilization) add (sterile) components listed in the table below depending on whether you want to make SOB or SOC.

Basic components SOB/SOC medium:

| Component | Amount | Final concentration |
| --- | --- | --- |
| Tryptone | 20 g | 2% |
| Yeast extract | 5 g | 0.5% |
| NaCl | 0.5 g | ~10 mM |
| 1M KCl | 2.5 mL | ~2.5 mM |
| dH2O | 800 mL | - |

SOB/SOC medium specific components:

| Component | SOB medium | SOC medium | Final concentration |
| --- | --- | --- | --- |
| 1M MgCl2 | 10 mL | 10 mL | ~10 mM |
| 1M MgSO4 | 10 mL | 10 mL | ~10 mM |
| 20% glucose | - | 18.018 mL | ~20 MM |

[Table of Contents](#table-of-contents)

## Chemically competent cells (E. coli DH5-ɑ + TG1)

In this protocol, you will find how to make chemically competent cells. Make sure to work on ice from step 3 onwards!

*Materials:*

* LB medium.
* 0.1 M CaCl2.
* Ice.
* ~86% glycerol.

*Protocol:*

1. Inoculate 50 mL LB medium with 0.5 mL overnight culture.
2. Grow the cells for approximately 2-3 hours at 37 ºC while shaking at 220 rpm until the OD600 is between 0.4-0.5. Take measurements each
   half hour.
3. Spin down cells at 4000 rpm for 10 minutes at 4 ºC. In all next steps: keep cells on ice.
4. Remove supernatant
5. Resuspend cell pellet in 25 mL ice-cold 0.1 M CaCl2.
6. Incubate cells on ice for 30 minutes.
7. Collect cell pellet by centrifugation at 4000 rpm for 10 minutes at 4 ºC.
8. Remove supernatant.
9. Carefully resuspend the cell pellet in 0.75 mL of 0.1 M CaCl2.
10. Add 129.31 µL ~86% glycerol.
11. Make aliquots of 100 µL and store cells at -80 ºC.

[Table of Contents](#table-of-contents)

## Electrocompetent cells (E. coli DH5-ɑ) and electroporation

In this protocol, you will find how to make electrocompetent cells.

*Materials:*

* LB medium.
* 10% glycerol.
* Ice.
* Selective plates with the corresponding antibiotics.

*Protocol (Competent Cells):*

1. Dilute 50 mL LB medium 1:50 with fresh overnight. of E. coli.
2. Follow the OD600.
3. At OD600 0.4-0.5, centrifuge at 8000 rpm, 4 ºC (from this moment: work as cold as possible).
4. Wash pellet successively with.
   * 40 mL ice-cold 10% glycerol.
   * 25 mL ice-cold 10% glycerol.
   * 10 mL ice-cold 10% glycerol.
5. Remove all supernatant and resuspend pellet in 100 µL ice-cold 10% glycerol.
6. Distribute aliquots of 50 µL in Eppendorf tubes, store at -80 ºC.

*Protocol (Electroporation):*

1. Cool the electroporation cuvette on ice.
2. Mix up to 5 µL DNA with 50 µL competent cells.
3. Add DNA/cell mix to the cuvette. Check that the cells are at the bottom of the cuvette and that it does not contain air bubbles.
4. Electroporate: 2.25 kV, 25 µF, 200 ohm.
5. Immediately afterwards: add 1 mL LB medium.
6. Incubate 1-1.5 hours at 37 ºC (preferably slow swirling).
7. Plate 100 µL dilutions on (selective) agar plates.
8. Incubate overnight at 37 ºC.

[Table of Contents](#table-of-contents)

## E. coli Transformation

In this protocol, you can find how to transform E. coli cells (how to insert the desired plasmid into E. coli) from competent E. coli cells
aliquots.

*Materials:*

* Ice.
* Prewarmed heat block at 42 ºC.
* Selective plates with the corresponding antibiotics.
* LB medium or SOC.
* Competent cells.
* Plasmid DNA.

*Protocol:*

1. Keep competent cells on ice when thawing.
2. Resuspend 5 µL of assembly product in 50 µL of cells in cold 1.5 mL Eppendorf tubes.
3. Leave cells on ice for 30 minutes.
4. Heat shock the cells for 30 seconds at 42 °C.
5. Leave cells on ice for 2 minutes.
6. Add 950 µL of SOC/LB.
7. Incubate at 37 °C in a shaker at ~220 rpm.
8. Spread 100-300 µL of cells onto (selective) agar plates.
9. Incubate the plates overnight at 37 °C.

[Table of Contents](#table-of-contents)

## Golden Gate Assembly Cloning using SapI

In this protocol, you can find how to perform a Golden Gate assembly protocol using SapI. This protocol is based on the New England BioLabs
Golden Gate Assembly Protocol using SapI.

*Materials:*

* T4 DNA Ligase.
* T4 DNA Ligase Buffer.
* SapI.
* Plasmid.
* DNA insert.
* MillQ.

*Protocol:*

1. Set up a 20 µL assembly reaction according to the table below.
2. Put the reaction mixture into a PCR machine for 1 hour at 37 °C.
3. Perform agarose gel electrophoresis or transformation to see whether the assembly was successful.

Components 20 µL Golden Gate assembly reaction:

| Components | 20 µL Reaction |
| --- | --- |
| Plasmid | 3 nM (final concentration) |
| Insert | 3 nM each insert (final concentration) |
| SapI 10 U/µL | 1.5 µL (15 units) |
| T4 DNA Ligase 2000 U/µL | 0.25 µL (500 units) |
| T4 DNA Ligase Buffer (10x) | 2 µL |
| MilliQ | To 20 µL |

[Table of Contents](#table-of-contents)

## Phusion Polymerase Chain Reaction (PCR)

In this protocol, you can find how to perform a Phusion polymerase chain reaction (PCR). This protocol is based on the New England BioLabs
PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530).

*Materials:*

* 5x Phusion HF or GC buffer.
* dNTPs.
* Template DNA.
* Forward and reverse primer for your template DNA.
* Phusion DNA Polymerase.
* MilliQ.

*Protocol:*

1. Prepare the PCR reaction mixture based on the composition described in the table below. If you want to run multiple PCR reactions, it is
   convenient to make a Master Mix consisting of the 5x Phusion buffer, dNTPs, template DNA, Phusion DNA Polymerase, and MilliQ. You can make
   a primer mixture in MilliQ with 10 µM of forward primer and 10 µM of reverse primer and use 5 µL of that per 50 µL PCR reaction. Add the
   primer mixture to the Master Mix right before putting the samples in the PCR machine.
2. Mix the total PCR reaction mixture in the PCR tube gently by pipetting up and down.
3. Insert PCR tubes in the PCR machine using the settings depicted in the figure below. Perform 25-35 cycles of the denaturation, annealing,
   and extension steps. The annealing temperature was calculated using the New England BioLabs (NEB) Tm calculator.

Composition Phusion PCR reaction mixture for 50 µL PCR reaction:

| Component | 50 µL Reaction | Final Concentration |
| --- | --- | --- |
| MilliQ | To 50 µL |  |
| 5x Phusion HF or GC buffer | 10 µL | 1x |
| 10 mM dNTPs | 1 µL | 200 µM |
| 10 µM Forward Primer | 2.5 µL | 0.5 µM |
| 10 µM Reverse Primer | 2.5 µL | 0.5 µM |
| Template DNA | Variable | < 250 ng |
| Phusion DNA Polymerase | 0.5 µL | 1.0 units/50 µL PCR |

Thermocycling conditions Phusion PCR:

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 °C | 30 seconds |  |
| Denaturation | 98 °C | 5-10 seconds | 25-35 cycles |
| Annealing | 45-72 °C | 10-30 seconds |
| Extension | 72 °C | 15-30 seconds per kb |
| Final extension | 72 °C | 5-10 minutes |  |
| Hold | 4-10 °C |  |  |

[Table of Contents](#table-of-contents)

## Q5 Polymerase Chain Reaction (PCR)

In this protocol, you can find how to perform a Q5 polymerase chain reaction (PCR). This protocol is based on the New England BioLabs PCR
Protocol for Q5® High-Fidelity 2X Master Mix.

*Materials:*

* Q5 High-Fidelity 2X Master Mix.
* Forward and reverse primer for your template DNA.
* Template DNA.
* MilliQ.

*Protocol:*

1. Prepare the PCR reaction mixture based on the composition described in the table below. If you want to run multiple PCR reactions, it is
   convenient to make a Master Mix consisting of the Q5 High-Fidelity 2X Master Mix, template DNA, and MilliQ. You can make a primer mixture
   in MilliQ with 10 µM of forward primer and 10 µM of reverse primer and use 5 µL of that per 50 µL PCR reaction. Add the primer mixture to
   the Master Mix right before putting the samples in the PCR machine. For plasmid DNA, it is recommended to use 1 pg to 10 ng of the
   template DNA per 50 µL reaction.
2. Mix the total PCR reaction mixture in the PCR tube gently by pipetting up and down.
3. Insert PCR tubes in the PCR machine using the settings depicted in the figure below. Perform 25-35 cycles of the denaturation, annealing,
   and extension steps. The annealing temperature was calculated using the New England BioLabs (NEB) Tm calculator.

Composition Q5 PCR reaction mixture for 50 µL PCR reaction:

| Component | 50 µL Reaction | Final Concentration |
| --- | --- | --- |
| MilliQ | To 50 µL |  |
| Q5 High-Fidelity 2X Master Mix | 25 µL | 1x |
| 10 µM Forward Primer | 2.5 µL | 0.5 µM |
| 10 µM Reverse Primer | 2.5 µL | 0.5 µM |
| Template DNA | Variable | < 1000 ng |

[Table of Contents](#table-of-contents)

## Colony PCR

In this protocol, you can find how to perform a colony polymerase chain reaction (PCR).

*Materials:*

* Incubated plate with colonies.
* Prewarmed heat block at 95 ºC.
* Forward + reverse primers for DNA plasmid.
* Phire PCR master mix.
* MilliQ.

*Protocol:*

1. Choose between 8-16 colonies of the incubated plate of interest (+1 negative control), depending on how many plates you have.
2. Mark one 1.5 mL Eppendorf tube for each colony with ‘A’ (sterile) and another 1.5 mL Eppendorf for each colony ‘B’ (non-sterile).
3. Add 25 µL of sterile MilliQ to all A Eppendorfs.
4. Resuspend colonies in A Eppendorfs.
5. Transfer 12.5 µL of sample to B Eppendorfs.
6. Heat shock B Eppendorfs for 5 minutes at 95 °C in heat block.
7. Let B Eppendorfs rest on ice for 5 minutes.
8. Centrifuge B Eppendorfs at max speed for 2 minutes.
9. Add 3 µL of sample from B Eppendorfs to PCR tubes.
10. Make a master mix of 1 µL (10 µM) of forward primer, 1 µL (10 µM) of reverse primer, 5 µL of MilliQ, and 10 µL of 2x Phire green hot start
    II PCR mix for each reaction.
11. Run the Phire PCR program according to the settings in the table below. The annealing temperature can be calculated using the Tm
    calculator from the 2x Phire green hot start II PCR mix supplier.
12. Run samples on agarose gel electrophoresis to see if the desired DNA fragments are obtained.
13. If the desired DNA plasmid was found in a specific colony, use the corresponding Eppendorf A of that colony to prepare an overnight
    culture.

Components colony PCR 20 µL Reaction:

| Component | 20 µL Reaction | Final concentration |
| --- | --- | --- |
| 2x Phire green hot start II PCR Mix | 10 µL | 1x |
| Forward primer | X µL | 0.5 µM |
| Reverse primer | X µL | 0.5 µM |
| MilliQ | 5 µL |  |
| Colony sample material | 3 µL |  |

Thermocycling conditions Phire PCR:

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 °C | 30 seconds |  |
| Denaturation | 98 °C | 5 seconds | 25-35 cycles |
| Annealing | X °C | 5 seconds |
| Extension | 72 °C | 10-15 seconds per kb |
| Final extension | 72 °C | 1 minute |  |
| Hold | 4 °C |  |  |

[Table of Contents](#table-of-contents)

## Restriction/Diagnostic Digest

In this protocol, you can find how to perform a digest. Digests can be done to prepare DNA fragments for molecular cloning or to identify
the identity of a plasmid.

*Materials:*

* Miniprep kit.
* DNA.
* 10x rCutSmart Buffer.
* Appropriate restriction enzymes.
* MilliQ.

*Protocol:*

1. Miniprep overnight cultures. For the miniprep, we used the QIAprep(R) Spin Miniprep Kit from QIAGEN.
2. Measure plasmid concentration with NanoDrop.
3. Based on the plasmid concentration, you will add certain volumes of Plasmid, and master mix (made up of MilliQ, buffer, and enzymes). If
   you only use one restriction enzyme, you can add 0.25 µL (diagnostic) or 0.5 µL (for cloning). The total volume should be 25 µL or 50 µL.
4. Incubate at 37 °C for 1 hour.
5. Perform agarose gel electrophoresis to analyze the results.

Composition restriction digest reaction mixture for 25 µL and 50 µL reaction:

| Components | 25 µL Reaction (Diagnostic) | 50 µL Reaction(For cloning) |
| --- | --- | --- |
| DNA | 1 µg | 1-2 µg |
| 10x rCutSmart Buffer | 2.5 µL | 5.0 µL |
| Enzyme 1 | 0.125 µL | 0.25 µL |
| Enzyme 2 | 0.125 µL | 0.25 µL |
| MilliQ | To 25 µL | To 50 µL |

[Table of Contents](#table-of-contents)

## Ligation

In this protocol, you can find how to perform a ligation reaction.

*Materials:*

* Insert DNA.
* Vector DNA.
* 10x T4 DNA Ligase Reaction Buffer.
* T4 DNA Ligase.
* MilliQ.

*Protocol:*

1. Use the New England BioLabs (NEB) Ligation Calculator to calculate how much vector (x) and insert (y) DNA is needed for an appropriate
   insert:vector ratio. The total volume of insert and vector DNA is a maximum of 17 µL.
2. Mix the components of the ligation reaction in a PCR tube in the order depicted in the table below (top to bottom). T4 DNA ligase should
   be added last!
3. Leave the ligation reaction for 1 hour at room temperature for the ligation reaction to occur.
4. Transform 5 µL of the reaction mixture into 50 µL competent cells to see whether the ligation reaction has worked.

Composition ligation reaction mixture for 20 µL ligation reaction:

| Component | 20 µL Reaction |
| --- | --- |
| T4 DNA Ligase Reaction Buffer | 2 µL |
| Vector DNA | (x) |
| Insert DNA | (y) |
| MilliQ | To 20 µL |
| T4 DNA Ligase | 1 µL |

[Table of Contents](#table-of-contents)

## Agarose Gel Electrophoresis

In this protocol, you can find how to perform agarose gel electrophoresis for the analysis of PCR results or digestion of plasmid results.

*Materials:*

* Agarose.
* TAE buffer (1x).
* Gel electrophoresis set-up.
* SERVA DNA Stain G.
* To make 1% agarose gel:
* 500 mL TEA buffer.
* 5g agarose.
* Melt in the microwave until the mixture is clear.

*Protocol:*

1. Prepare the gel: Mix 40-50 mL of agarose gel mix (we used both 0.9% and 2%) with 0.8 µL dye (always use gloves). Pour the solution into
   the gel cast and insert combs. Wait for solidification (~20-40 minutes).
2. Mix the prepared sample with loading dye (Gel Loading Dye Purple (6x), NEB) (5:1 ratio, so for example 5 µL sample and 1 µL loading dye),
   and load a total volume of 5 µL or 10 µL on the gel. If the dye is already mixed in the sample (e.g. as for a colony PCR), pipette 5-10 µL
   of the samples directly into the well.
3. Add 5 µL of ladder (Quick-Load Purple 1 kb Plus DNA Ladder, NEB) to one of the wells (either first or last is convenient for analyzing the
   gel).
4. Run the gel at a constant 120 V for 30-40 minutes.
5. Analyze the gel using a GelDoc (Bio-Rad) imager.

[Table of Contents](#table-of-contents)

## Sequencing

In this protocol, you can find how to prepare samples for sequencing.

*Materials:*

* Miniprep kit.
* 1.5 mL Eppendorf tubes.
* Overnight culture of bacteria containing plasmid.

*Protocol:*

1. Miniprep the overnight samples. For the miniprep, we used the QIAprep(R) Spin Miniprep Kit from QIAGEN.
2. Transfer 5 µL of the sample to a clean 1.5 mL Eppendorf and add 5 µL of 10 µM forward primer.
3. Transfer 5 µL of the sample to a (separate) clean 1.5 Eppendorf and add 5 µL of 10 µM reverse primer.
4. Label each of the Eppendorf tubes and send for sequencing.

[Table of Contents](#table-of-contents)

## Antibiotics concentrations

While working in the lab, we used different antibiotics for selection of our plasmids in E. coli. Here you can find an overview of the stock
and working solutions.

*Ampicillin:*

* Stock solution: 100 mg/mL.
* Working solution: 100 µg/mL.
* Dilution factor: 1000.
* Results in: 1 µL per mL media.

*Chloramphenicol::*

* Stock solution: or 25 mg/mL.
* Working solution: 25 µg/mL.
* Dilution factor: 1000 .
* Results in: 1 µL per mL media.

*Kanamycin:*

* Stock solution: 50 mg/mL.
* Working solution: 50 µg/mL.
* Dilution factor:1000.
* Results in: 1 µL per mL media.

[Table of Contents](#table-of-contents)

## GFP Isolation

In this protocol, you can find how we isolated GFP to use for measurements with our electronic device.

*Materials:*

* Overnight culture of bacteria containing GFP plasmid.
* LB medium.
* Y-PER.

*Protocol:*

1. Transfer 0.5 mL of an overnight culture containing the plasmid with GFP to 50 mL fresh LB medium.
2. Grow culture to OD = ~1.0.
3. Spin down cells.
4. Resuspend in 200 µL Y-PER.
5. Incubate cells for 20 minutes.
6. Spin down cells in a tabletop centrifuge for 20 minutes at full speed.
7. Collect supernatant and store in ~-20 °C freezer.

[Table of Contents](#table-of-contents)

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