iGEM 2023 — Team tec-chihuahua

# Notebook

### Chronological Notes of our Team's Work

* **13th:** The paperwork of those interested in being part of the iGEM Tec-Chihuahua 2023 team began to be received.
* **17th:** The call to join the team was closed.
* **20th:** The pre-selection results were published.
* **21st, 2nd, and 23rd:** Interviews were conducted with each of the preselection candidates.
* **28th:** The final list of those selected to make up the iGEM Tec-Chihuahua 2023 team was published.

* **1st:** The team members were called to take the official photo.
* **6th:** Friendly meeting in a restaurant in the city where the team members introduced themselves with each other.
* **7th:** First official meeting of the team together with PI, advisors, and collaborators. The official start of activities took place.
* **17th:** Generalities of the competition and the team were described. The logistics of the upcoming events were agreed upon and 4 teams were assembled to present the first ideas of possible projects to compete in iGEM 2023.
* **24th:** The team met to prepare for the first fundraising activity. The creation of posters.
* **25th:** Fundraising activities began.
   Adolfo, Arely, Vannessa and Giuliana attended the SEDENA event (by its acronym in Spanish: Secretariat of National Defense) representing the iGEM project in Chihuahua.
* **31st:** The first feedback session was held on the proposals made by each of the 4 teams.

* **9th:** Baking brownies in one of the members' house for the later sale as a fundraising activity.
* **24th:** Charlottes were baked by Sofi and the following days they were sold by the team members as a fundraising activity.

* **6th:** Team members met to select area leaders and team leader.
* **12th:** The final presentation of proposals was made to experts and voting was carried out selecting 1 of the four projects. The selected proposal would be the one that will be developed to compete in iGEM 2023.
* **18th:**We finally selected one of our project ideas after a meticulous review with our instructors and various experts on each area. Also, we structured our team areas and leaders.

* **7th:** The project to be developed for iGEM was changed to the second proposed option.
* **22nd:**  The initial version of the Safety form was uploaded.
* **24th:**  Creation and update of the lab notebook.
* **28th:** The team decided the village selection.
* **29th:** A draft version of project description was uploaded in the team wiki.
* **30th:** Interview with José Manuel Oropeza.

* **2nd:** The context of the problem of mastitis was thoroughly investigated.
* **3rd:** Zoom interview with Imelda Ramos.
* **5th** Visit to DELMAR.
* **7th:** The human, entrepreneurship and wiki team visited the Zaragoza dairy farm.
* **10th:** *In silico* simulation of constructs in agarose gel electrophoresis.
   Resuspension of LysCSA13 and LysK DNA from Twist.
   The impact grant call was reviewed.
   CBTa 90 was visited in Cuauhtemoc to learn the milking process.
   Class taught by Adrian Sandoval on mastitis.
* **11th:** Production of chemocompetent 5-alpha cells.
   Transformation of pUC19 with chemocompetent cells.
   Start competitor analysis for AureoBos.
   The sponsorship document was sent to Los Pinos.
* **13th:** Surveys were conducted with dairy consumers in our community and an informative brochure was distributed to them.
* **15th:** The interview was carried out with the UACJ (for its acronym in English, Autonomous University of Ciudad Juarez).
* **15th and 16th:** Attend the iGEM Entrepreneurship Summer School.
* **17th:** Visit to Establo 1967 in Meoqui, Chihuahua, Mexico.
   Visit to the cowshed called 1967.
* **18th:** Golden Gate assembly of LysCSA13 and LysK transcriptional units.
   Visit to Cesar Loya's small cowshed.
* **19th:** Interview with Eng. Betty Castro.
* **24th:** Register AureoBos in Stella Orion.
   Sponsorships were requested to local and national laboratories.
* **27th:** Writing of Manual 101 (contribution by entrepreneurship).
* **28th:** Visit to Mennonite Dairy.

* **2nd:** SDS-PAGE run of LysCSA13 and LysK.
   LysCSA13 and LysK protein induction kinetics.
   Interview with ECSG (for its acronym in English for School of Social Sciences and Government).
* **4th:** Interview with Veterinary Pharmaceutical called Zoetis.
   Reactivation of *S. aureus*.
* **7th:** Collaboration with the iGEM niSkin team from Virginia.
* **10th:** The AureoBos product was classified as biological.
* **14th:** Resuspension of LysSS DNA from Twist.
* **16th:** Interview with Dr. Francisca Villanueva.
* **19th:** LysSS protein induction kinetics.
   Digestion of LysK, LysCSA13 and LysSS with SmaI enzyme.
* **25th:** The team went to take a group photo at the DELMAR farm in Delcias, Chihuahua, Mexico.
* **28th:** Zoom meeting with the animal health pharmaceutical MSD specifically with Pepé. Where we validated our way of commercializing.
   Digestion of LysK, LysCSA13 and LysSS with EcoRI enzyme.
* **30th:** Leopoldo Escarcega, an entrepreneurship intern, was added to attributions.
* **31st:** The team bought the Grand Jamboree tickets.
  The results of the Innovation Awards by Universidad Panamericana (UPIA) were announced.
   The congress called expoagro was attended.

* **5th:** Arely met with the director of our Tec-Chihuahua campus, Armando Bahena to explain our project.
* **6th:** Intellectual Property Session with Feli Sandoval, a member of the Entrepreneurship Ecosystem and the Technology Transfer Office at our university.
   Reactivation of *S. agalactiae* donated by the National School of Biological Sciences.
* **7th:** First pitch at Startup Chihuahua at the conference “Making the Invisible Visible”.
* **8th:** The team roaster was modified.
  The summary and title of the project were added to the iGEM page.
* **9th:** Working sessions with team to reflect progress on wiki.
* **12th:** Second pitch in the framework of Semana Tec.
  Visit to Rancho La Campera.
* **13th** The team's project promotion video was uploaded.
* **14th:** Team rosters freeze.
  Pitch feedback session with entrepreneurship ecosystem.
  Meditation session with the team.
* **15th:** CONECTA meeting note.
* **20th** Attendance at DIGAL (for its acronym in Spanish: International Dairy Farmer's Day) in Delcias, Chihuahua
* **21st:** Final Project Safety form was uploaded.
  Pitch Night Ilum.
* **22nd** Attendance at DIGAL (for its acronym in Spanish: International Dairy Farmer's Day) in Delicias, Chihuahua

* **5th: UP Innovation Awards**
Topi and Arely went to Ciudad de México to participate in the final division of this competition. After some pitches and conferences, AureoBos ended up in first place of the cathegory of best validated idea.* **9th: Stakeholder meeting:** Team Human had a meeting with nanoparticles expert Armando Zaragoza.
* **12th: Wikifreeze:** All the team worked hard pouring our project into this wiki. This was the end of it.

![](https://static.igem.wiki/teams/4607/wiki/eng/img-9762-1.webp)

# Engineering

# From DNA to impact, we engineer solutions for our world.

## Overview

* [ ]
  Design

  Use Engineering principles to describe a biological system in an intended model, to improve our
  initial design, or its posterior modifications, favoring the product development. It must consider
  what the system should or should not do, and what's vital to the successful product usage.
* [ ]
  Build

  Describe the process that we previously proposed, highlighting its qualities and requirements.
  This stage usually includes model considerations, and prototype simulation to pre-aboard testing.
* [ ]
  Test

  Test the design built to compare results with the expected outcome simulated.
* [ ]
  Learn

  Identify the main issues and discrepancies between the expected results and the real evidence,
  denoting the bottleneck and focusing on the main mistakes.
* [ ]
  Reflection

  Closes the cycle by applying the knowlede acquired through our process.

 ![](https://static.igem.wiki/teams/4607/wiki/eng/success.png)

This page takes you along AureoBos’ journey by capturing the trial and error occurred during the project
and how we solved the challenges that came up through iterations of the engineering cycle. By applying this
method, we integrate all the stakeholders’ suggestions to obtain an intended product that responds to the
world's necessities.

Any engineering project involves things that do not go right the first time, and while this can be
daunting, you can learn and adjust details to make it work based on mistakes or blind spots. Considering the
**iGEM Engineering Cycle** and its four phases, our product was developed by **three great cycles**,
**ten semi-cycles**, and **13 iterations**.
Also,we decided to complement our stages by adding semi-cycles and a fifth stage: **Reflection**, which
closes the cycle by representing the way to apply the knowledge acquired during all the process, associating
it with a complete new iteration cycle. It formulates a **new question** to address, along with the
corresponding **method to resolve it**. In the same way, it makes possible the extrapolation of knowledge
to prevent mistakes.

## Main cycles

Click "Go to section" to change between sections

Protein
 Induction

Go to section

Golden Gate Assembly

Go to section

![](https://static.igem.wiki/teams/4607/wiki/eng/taffy-engi-sf.png)

First sub-cycle:

Second sub-cycle

Third sub-cycle:

## First Cycle: DNA Assembly

Our product is composed of three fusion-endolysins: [LysCSA13, LysK, and LysSS](parts). The aim of the Golden Gate Assembly cycle was to
obtain our expression constructs.

### First sub-cycle: Chemocompetence and transformation

Click on
the
accordions to see details about each iteration

1st Iteration

Design

Through a [sponsorship](https://2023.igem.wiki/tec-chihuahua/sponsors) we
received *Escherichia coli 5-alpha* competent cells. After using them all we performed a bacterial reactivation to conserve
the
cell strain, which was very useful for us in the Golden Gate Assembly phase. We performed a
chemocompetent cell’s production to transform our *E. coli* strains (5-alpha, and
BL21(DE3)) for the storage of our constructs and its posterior expression.
After inoculating the *Escherichia coli 5-alpha* strain in a LB agar plate, its competent
ability got lost. To use them in the transformation we followed the protocols for chemically
competent cells and transformation used by [Tec-Chihuahua 2022](https://2023.igem.wiki/tec-chihuahua/experiments).

Build

An individual *E. coli 5-alpha* colony was used to inoculate LB culture
medium in order to do the protocols of forming a cell pellet. To continue with the
transformation
process, we made 50 uL aliquots to add 5 µL of the Golden Gate Assembly plasmid DNA, and mixed
by
inverting 5 times. After ending with the protocol, we incubated overnight at 37°C.

Test

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/tranformacion-0.jpg)

**Figure 1**. First iteration’s transformation results.

The *E. coli 5-alpha* chemically competent cells were transformed with a
control plasmid, pUC19, to prove that there was a good transformation efficiency. But nothing
meaningful has grown. In the same way, the reaching of the optical density was much longer than
that we expected, about nine times more than the 30 minutes described in the protocol. We knew
that cells survived the heat shock of the transformation process, because our competent cell
control showed cell growth, but we couldn’t know if the complication was in the competent cell
protocol or in the transformation one.

Learn

The efficiency of transformation was very low. We assumed that it might be due
to rough manipulation of the cells, that the CaCl2 for the cells was not very cold and that the
OD
was barely between the appropriate range of 0.6 to 0.8. Besides, we considered that could be
related to the heat shock temperature, because of the poor water bath thermostat definition.

Reflection

We have evaluated the learning stage, consequently we identify that the apparent
issues were about the cell’s manipulation. The new question was related to the temperature
required in the process, and how we could be completely assured of their values. In the same
way,
we have evaluated the incubation times, where we decided to increase their lapses. We conducted
an
in deep-analysis of the entire process, and discovered that the shaking step wasn't as efficient
as it should be. To solve these problems, we decided to analyze the original protocols and
suggestions of the NEB website, and review troubleshooting data sheets.

2nd Iteration

Design

Considering our past observations, we found that some of the most common complications in the
cell transformation are related to the heat shock temperature, in which an unstable control of
the ranges hinder the process. In the same way, the cold temperatures are very important in the
cell competent protocol, including the material and consumables used. We also considered the
option of using our -80°C stored cryovials.

Build

To perform our second iteration we modified the chemocompetent protocol described in the Experiments
Page of the Tec-Chihuahua 2022 Team, assuring that all the equipment required was at 4°C,
and all the material and consumables were previously put on ice. In the same way, we have
ensured that our water bath was at 42°C by double-checking the temperature with an additional
termometer. Finally, we increased the incubation time up to 2 hours, realizing that Eppendorfs
position was actually allowing the culture movement.

Test

Our results were increased in comparison to our first iteration, but they were still lower in
comparison between NEB cell competent colony forming units. At this time we have positive
results to our transformation protocol as well as our cell competent control, but no meaningful
result has been achieved for the cryovials samples.

Learn

We actually increased the colony forming units of our transformation protocol, in which we
assumed that was related to the assuring of the temperature of the water bath in the
transformation protocol, as well as the constancy in the cold temperature of the entire
chemocompetent cell process. We also achieved that zero transformation efficiency was obtained
from the cryovials previously made, hence we considered that it is crucial to perform a new
chemocompetent cells protocol by the time that transformation is required. Although we improved
our results by using the Engineering cycle, we considered that these results were not good
enough to try with our Golden Gate Assembly construct.

Reflection

We realized that by using the Engineering Cycle was possible to upgrade our results in the
number of colony forming units of our transformation protocol, however, we weren’t safe enough
to take the risk trying cells' transformation with our Golden Gate Assembly construct, because
is well-know that transforming with a generic positive control achieve better yields that which
are obtained with other samples.
To continue improving our results we analyzed every step of the previously mentioned protocols,
evaluating the possibility to increase the cell concentration plated over the agar plate without
risking the absorption of bacteria, besides considering the optimization of the times’ process.
The next question was related to the optimization of our process, and the solution was planned
to appear by investigating the past iGEM Teams’ protocols.

3rd Iteration

Design

Evaluating the reflection of the past iteration we found the following options to optimize the
efficiency and time of the process. By increasing the available gas exchange surface we could
shorten the period to reach optimal density, thanks to its effect on accelerating cell
duplication. While it is possible to increase the efficiency of the transformation by
considering the concentration of cells plated on the agar medium, where it is possible to
increase the total number of cells by harvesting the pellet of transforming cells, and
resuspending it in a smaller volume.

Build

To perform our third iteration we modified the chemocompetent protocol described in the [Experiments
Page](https://2023.igem.wiki/tec-chihuahua/experiments) of the Tec-Chihuahua 2022 Team, applying the changes described in the second
iteration, and using a 250 mL Erlenmeyer flask instead a 100 mL one. In addition, its iteration
includes the harvest of the total transforming cells incubated for two hours, and resuspending
it into 100 uL SOC medium, to plated the total volume into preheated plates.

Test

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/tranformacion-final.jpg)

**Figure 2**. Third iteration’s transformation results.

Our results were up to the NEB cell competent efficiency, where the positive pUC-19 control
reached uncountable colony forming units. At the same time, the lapse to achieve the optical
density required for the competent cell protocol was shortened to ninety minutes.

Learn

We reached the NEB cell competent efficiency by incorporating all the modifications which
optimized the process, it was good enough to transform our Golden Gate Assembly constructs. Our
final protocols to the chemocompetent cell’s production, and heat-shock transformation are
available in our
[Experiments
Page](https://2023.igem.wiki/tec-chihuahua/experiments).

Success

We actually achieved good enough results, and obtained the standardization of our protocols to
continue with the process!

### Second sub-cycle: Golden Gate Assembly

Three fusion-endolysins are the nucleus of AureBos, our non-antibiotic treatment against the mainly
pathogenic bacteria which causes bovine mastitis: *Staphylococcus aureus*, *Streptococcus
agalactie*, *Streptococcus uberis*, and *Escherichia coli*. The biologic principle
behind our product has basis in the composition of the endolysins in which we conducted an in-deep
analysis to optimize the original capacities of these enzymes; eventually we synthesized our new
codifying sequences. All the endolysins principle is acutely described in our [Parts Page](https://2023.igem.wiki/tec-chihuahua/parts). As a
brief description each
fusion-endolysin is composed by three mainly domains: CHAP, in which lies the catalytic activity that
makes possible the lysate of specific pathogenic bacteria; the albumin binding domain (ABD) which
increase up to 30 hours the endolysin’s lifespan; and finally the SH3 domain, that gave to AureoBos
the biological safety through the ability to recognize specific bonds in the peptidoglycan layer of
the previously described bacterium. Attending the stakeholder’s suggestions, we expanded the effect
spectrum by the addition of a third endolysin which incorporates a polycationic nonapeptide, and the
Cecropin A peptide, both have abilities to destabilize the gram-negative bacterial cell wall. You
could know more about this incorporation in our [Human Practices Page](https://2023.igem.wiki/tec-chihuahua/human-practices).

Click on each
accordion to see details

1st Iteration

Design

To obtain our constructs we designed the codifying sequences for each endolysin as a result of
an extensive bibliographic search. After an in-depth research, we designed the endolysins that
will be expressed, LysCSA13 and LysK, with some modifications: an ABD and the cell-wall binding
domain (CBD) of SH3B30. The ABD increases the *in vivo* half-life from 20 minutes to 30
hours2 and the CBD makes the endolysin specific for *Staphylococcus aureus*,
*Streptococcus agalactiae* and *Streptococcus uberis*.3, 4 A third
endolysin (PCNP-CecA-LysSS) was added as the way to incorporate the stakeholders’ suggestions.
Both enzymes were modeled in AlphaFold 2, to verify the proper folding of the protein. In the
case of the LysK endolysin, the cleavage site was not necessary since the addition of the
histidine tag did not interfere with the folding of the enzyme. The sequences were optimized for
*E. coli* and synthesized as a transcriptional unit with a sponsorship from TWIST. The
biobricks were selected and assembled *in silico* using Snapgene. For the constructions
of both enzymes, the same parts were used: promoter (BBa\_J435350), RBS (BBa\_Z0262), each coding
sequence, histidine tag, terminator (BBa\_J435371) and backbone (BBa\_J435330) were assembled by
Golden Gate (Figure 3).

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/todas-las-endo.jpg)
**Figure 3**. Expressions cassettes for the endolysins LysCSA13-ABD (a), LysK-ABD-SH3B30 (b).
and PCNP-CecA-LysSS (c)

Build

To perform the Golden Gate Assembly cloning method we followed the NEB’s protocol, attending all
the advice from the reagent’s trading company. All the process is described in our [Experiments Page](https://2023.igem.wiki/tec-chihuahua/experiments). We resuspended
the DNA of the backbone from the plate with nuclease-free water. The DNA was transformed into E.
coli DH5α from NEB and extracted with the Miniprep protocol.
When we received the DNA of the transcriptional units from TWIST, we resuspended and stored it.
The Golden Gate assembly was performed with enzyme BsaI, the backbone, and the synthesized
transcriptional unit. Three mixes of the reaction were made, one for each transcriptional unit,
LysK, LysCSA13, and LysSS. A negative control was made for each reaction. Posterior to the
transformation with our constructs, we planed the plasmid DNA extraction resulting of a Miniprep
reaction, and finally, evaluating the plasmid concentration, plasmid topology, and weight to
compare with our *in silico* digestions, to assured that we had our endolysin constructs.

Test

The constructs were transformed into *E. coli 5-alpha* by heat shock and inoculated in
kanamycin plates. We obtained individual colonies in every plate, including the negative
controls, in which nothing should have grown.

Learn

When analyzing our results, we remember that (Figure 4) the chosen backbone contains two
resistances to antibiotics (kanamycin and ampicillin), after being cut in the Golden Gate
reaction, it only retains the resistance to kanamycin.1 However, We realized that there was no
way to have 100% efficiency in the cloning stage, and considering that we didn't add any
fluorescence or color reporter genes, we were in a hard situation. In other words, we could not
differentiate which colonies were transformed with our endolysin constructs, and which colonies
were transformed with the original backbone with both resistances.

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/backbonevectorsnpgn.png)
**Figure 4**. Backbone after Golden Gate assembly.

Reflection

Evaluating the situation, we planned to identify our constructs by using all the information
about our current plasmids. The new challenge was to find the way to counteract the reporter
gene absence, with the knowledge of having two resistance genes in the undigested acceptor
backbone, and only one resistance in our interest plasmids. We also confirmed that the
transformation efficiency was very positive.

2nd Iteration

Design

After reflecting on our mistakes, we realized that it was possible to identify endolysin
constructs by inoculating the same colony onto kanamycin and ampicillin plates. We reached
almost 90 transformed colonies and considered 25 colonies to be significant to increase the
chances of choosing the right transformant.

Build

We chose 25 different colonies from every endolysin to inoculate in kanamycin and ampicillin
plates. Later, we designed the work plan in which we divided each plate into four quadrants, in
the same way, all the labeling was made to duplicate, one by each antibiotic. After all the
experiment was correctly labeled, we performed four different cross-striations by each plate,
identifying every colony by a number, which was the same for each antibiotic. Finally, we
incubated all the plates at 37°C for 18 hours. To confirm that our endolysin constructions were
in our plates, we expected that the growth would appear only in the kanamycin plates, but
nothing in the ampicillin ones. While the growth on both of the plates would reveal an acceptor
backbone transformant.

Test

We showed bacterial growth in both plates for the majority of the colonies, but some of the
colony samples appeared only in the kanamycin plates (Figure 5).
![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/colonias16y18lysk-3.png)

**Figure 5**. LB agar plates with kanamycin (50 g/mL) from the
transformation of the LysK construct. A. Transformed *E. coli 5-alpha* with the plasmid
of LysK (quadrant 16). B. Transformed *E. coli 5-alpha* with the plasmid of LysK
(quadrant 18).

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/gel-de-transformantes.jpg)

**Figure 6** LB agar plates from the transformation of the LysK construct.
A. LB agar with ampicillin (100 g/mL) inoculated with transformed *E. coli 5-alpha* with
the plasmid of LysK (quadrant 18). B. LB agar with kanamycin (50 g/mL) inoculated with
transformed *E. coli 5-alpha* with the plasmid of LysK (quadrant 18).

Learn

We found our endolysin constructs!
The antibiotic screening made possible the identification of our endolysin constructions,
confirmed by the growth of the transformed bacteria only in the kanamycin plates. We also have
growth of some of the colonies in both of the plates, which corresponded to the acceptor
backbone transformants. Any of the colonies grow only in the ampicillin plates, which coincided
with our expectations.

Reflection

We assumed the Golden Gate reaction was done correctly, due to the fact that growth was only
present on the kanamycin agar plates for some of the replated colonies. Otherwise, it would have
grown in the presence of both antibiotics, kanamycin and ampicillin, meaning that the backbone
was still intact.
When performing a ligation reaction of any kind, the backbone can get auto-ligated. This occurs
because of the ligases that are not specific to a site, unlike enzymes, which make them very
prone to ligate the same backbone without the insert. In a first instance, there is nothing to
tell us that this did not happen without doing other types of tests 5. We inferred that some
colonies grew on the negative control because the backbone ligated itself without the
transcriptional unit; this was verified by the antibiotic selection mentioned above, confirming
that the colonies had the DNA of the backbone with both antibiotic resistances.
It’s very important to consider the addition of reporter genes for the construction of
expression plasmids, however, it is more important to evaluate the challenging situations by the
Engineering cycle, taking into account all the information that already exists. In the same way,
we conclude that the antibiotic screening is not enough to ensure the presence of our endolysins
constructs. At this point, our next step is to perform a Miniprep reaction of the supposed
transformants to obtain its plasmids and analyze them by an enzymatic digestion on an agarose
gel.

### Third sub-cycle: Golden Gate Assembly

The next sub-cycle had the objective to demonstrate our constructs identity to be completely assured
that we were obtaining the endolysins expression constructs, before performing the protein inductions.
In the same way, we had to extract the plasmids to their posterior heat shock transformation in the
*E. coli BL21 (DE3)* strain, which is feasible after the standardization of the protocols as we
did in the previous semi-cycles.

Click on each
accordion to see details

1st Iteration

Design

To continue with the experimentation, we planned to perform a plasmid DNA extraction for each of
the three endolysins’ constructs that compounds the AureBos treatment. Performing a plasmid DNA
extraction was possible after following the Miniprep PureLinkTm Quick protocol, which is also
included in the Experiments Page. In the same way, an agarose electrophoresis was required for
its semi-cycle.

Build

The building up of this stage was prepared considering all the steps described above, but now
taking special care of all the temperature steps, as we reflect in our previous semi-cycle
iterations. Our samples were the  *E. coli 5-alpha* apparently transformed with each of
the fusion-endolysins constructs inoculums, incubated at 37°C at the maximum shaking parameters,
for 24 h.
For the plasmid analysis we performed an *in silico* digestion on the SnapGene software,
taking into account our fusion-ensolysins constructs, and the specific enzyme cuts: SmaI. Our
plasmids have a length of: 2997 bp (LysK), 3159 bp (LysCSA13), 2892 bp (LysSS). The *in
silico* results demonstrated the expected length for each linearization on a 1% agarose
electrophoresis. In Figure 7 is shown the expected results for LysK and LysCSA13, in which were
also included the acceptor backbone control, and the undigested samples for both, acceptor
backbone, and LyK construct.
![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/engin-fig-7.jpg)

**Figure 7**. *In silico* agarose gel (1%) of the results from the
enzymatic digestion with SmaI. MW. 1 kb DNA *Ladder 1*. Digested backbone (3893 bp).
*Ladder 2*. Negative control reaction of the backbone (3893 bp). *Ladder 3*.
Digested plasmid with LysK (2997 bp). *Ladder 4*. Negative control reaction of the
plasmid with LysK (2997 bp). *Ladder 5*. Digested plasmid with LysCSA13 (3159 bp).
*Ladder 6*. Negative control reaction of the plasmid with LysCSA13 (3159 bp).

Test

After the plasmids digestion, we performed an 1% agarose gel electrophoresis as is described in
our [Experiments Page](https://2023.igem.wiki/tec-chihuahua/experiments), to analyze the
plasmid identity of the LysK and LysCSA13 fusion-endolysin constructs. Our samples were
performed as Figure 8 reveals.

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/engin-fig-8.jpg)

**Figure 8**. *In silico* agarose gel (1%) of the results from the
enzymatic digestion with SmaI. *Ladder 1*. Quick-Load Purple 1 kb Plus DNA. *Ladder
2*. Digested backbone above 4000 bp (expected band size: 3893 bp). *Ladder 3*.
Negative control reaction of the backbone (3893 bp). *Ladder 4*. Digested plasmid with
LysK (2997 bp). *Ladder 5*. Negative control reaction of the plasmid with LysK, between
2000 and 3000 bp (expected band size: 2997bp). *Ladder 6*. Digested plasmid with
LysCSA13, two bands at 1200 bp and between 1500 and 2000 bp (expected band size: 3159 bp).
*Ladder 7*. Negative control reaction of the plasmid with LysCSA13, between 2000 and 3000
bp (expected band size: 3159 bp).

Learn

We realized that the gel's appearance was not the best, not only because of the undesired spots,
but also because the sample bands were not aligned to the DNA marker. In addition, an unexpected
double digestion was revealed for Ladder 6. After analyzing the entire experimentation, we found
that everything was according to the protocol, the DNA sequences were correctly reviewed, and no
problem was found in the *in silico* results. Evaluating the situation, we concluded that
there was a problem with the reagents because of their expiration date. To confirm our
hypothesis, we repeated the experiment, but nothing changed.

Reflection

To apply the learning from this iteration, we realized that some of our lab reagents were not in
good enough condition to be used for experimentation. Initially, we thought that this situation
would apply to the agarose, the staining gel’s SYBR Safe, but after looking for troubleshooting
sheets, we realized that the unexpected gel stains could be an incorrect manipulation while
preparing it. In the same way, we evaluated the possibility of having star activity as a
consequence of rough manipulation, but it could only occur during long incubation periods, which
was not the case. Our new question was related to finding a way to correctly evaluate the
plasmids' identity with a high level of confidence. To solve this problem, we consider the
option of using other enzymes and take into account the troubleshooting data sheets for
obtaining better agarose gels.

2nd Iteration

Design

After reflecting on our learnings we intended an analysis of the most common troubleshooting
data sheets related to the plasmid identification techniques, in which we realized that we could
improve our results by taking care of the agarose dissolving steps. In the same way, we
recognized another enzyme currently in our lab, which also produces plasmid linearizations,
hence we decided to repeat the experiment by using EcoRI for LysCSA13 and LysSS instead of SmaI,
which corresponded to the failed results. Finally, our new ladder arrived just in time to try
again, considering that some of the possible causes of the non alignment between the DNA ladder
and our samples could be because of the previous uses of this shared lab reagent.

Build

To perform this iteration we followed the current protocol described in our Experiments Page, in
which we consider a long time and less temperature to the dissolution process of the agarose.
Besides, we chose the EcoRI enzyme because it was also useful for our plasmids in producing pDNA
linearized. To be assured that it was a correct selection, we performed an *in silico*
digestion through SnapGene as is shown in Figure 9. At the same time, we incorporated the new
Quick-Load Purple 1 kb Plus DNA ladder.

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/gel-imageb.png)

**Figure 9.** *in silico* pDNA agarose gel electrophoresis (1%). (a)
*Lane 1.* Quick-Load® Purple 1 kb Plus DNA Ladder. *Lane 2.* LysK digested with
single-cut enzyme SmaI (2997 bp) pDNA.*Lane 3.* Negative control LysK digestion (2997 bp)
pDNA. (b) *Lane 4.*LysCSA13 digested with single-cut enzyme EcoRI. *Lane 5.*
Negative control LysCSA13 digestion (3159 bp) pDNA. (c) *Lane 6.* LysSS digested with
single-cut enzyme EcoRI (2892 bp).*Lane 7.* Negative control LysSS digestion (2892 bp)
pDNA.

Test

After running our 1% agarose electrophoresis we showed that our digestions coincided with the
*in silico* results expected (Figure 10).

![todas-las-agarosas](https://static.igem.wiki/teams/4607/wiki/parts/agarosas/todas-las-agarosas.jpg)

**Figure 10.** pDNA agarose gel electrophoresis (1%). (a) *Lane 1.*
Quick-Load® Purple 1 kb Plus DNA Ladder. *Lane 2.* LysK digested with single-cut enzyme
SmaI (2997 bp) pDNA.*Lane 3.* Negative control LysK digestion (2997 bp) pDNA. (b)
*Lane 4.*. Quick-Load® Purple 1 kb Plus DNA Ladder. *Lane 5.* LysCSA13 digested
with single-cut enzyme EcoRI. *Lane 6.* Negative control LysCSA13 digestion (3159 bp)
pDNA. (c) *Lane 7.* LysSS digested with single-cut enzyme EcoRI (2892 bp).*Lane
8.* Negative control LysSS digestion (2892 bp) pDNA. *Lane 9.* Quick-Load® Purple 1
kb Plus DNA Ladder

Learn

Once we analyzed our results, we discovered that using the enzyme EcoRI, it was possible to
linearize our plasmids, which gave us the possibility to compare our results with the expected
bands. In fact, we verified that our constructs were correctly assembled, besides, we improved
the agarose gel’s quality so that unwanted spots didn’t appear.

Reflection

The overall knowledge acquired for this iteration was related to the reagents' usage. Through
our first iteration, we faced a challenge because some of our plasmids weren’t correctly
digested, instead, a double digestion occurred, which had no relation to our designing or
assembly method; We confirmed it once our constructions were digested with another enzyme. The
only variation between our constructions was the codifying sequences, and considering that the
restriction site was in the acceptor backbone, no change should have shown between the plasmids’
digestions, but only the LysK plasmid aligned its results with the expected ones. After
performing the same experiment but using the EcoRI enzyme, all the results coincided with the
*in silico* simulation. Evaluating the situation and taking into account that the 1 kb
DNA ladder condition was not the best, we concluded that the obstacle was a consequence of
non-ideal storage conditions.

First sub-cycle:

Second sub-cycle

## Protein Induction

### First sub-cycle: Induction Math Model

1st Iteration

Design

Once we finalized the first cycle, we aimed to establish the conditions that would be most
favorable for protein induction. The gene expression system in *E. coli* BL21(DE3) is inducible
by isopropyl β-D-1-thiogalactopyranoside (IPTG), so our objective was to predict the protein
yield based on different initial IPTG concentrations and analyze how the system behaves while
accounting for stochasticity.

Build

The model was constructed by integrating the biochemical reactions within the cell,
encompassing the introduction of IPTG via simple diffusion. A representation of each binding
interaction was diagrammed, culminating in the development of a systematic set of Ordinary
Differential Equations (ODEs). The equations were derived in accordance with the Law of Mass
Action, ensuring a foundation for the subsequent analyses conducted in the model.
Additionally, we incorporated a range of parameters obtained from literature, accompanied by
the necessary assumptions to ensure model´s tractability.

Test

In addressing the system ́s behavior, we utilized a two-fold approach: solving the ODEs set
and performing a Monte Carlo simulation. The simulation was executed by incorporating a
defined normal distribution of known parameters, enabling an assessment of the stochasticity
of biomolecular mechanisms. However, it was not feasible to simultaneously plot each scenario.

Learn

During the analysis of this conflict, we identified a key factor to the numerical solver we
employed in MATLAB, the ODE15s. This solver dynamically adjusted steps to accurately
approximate the solutions based on the system´s evolving requirements. However, this led to
varying matrix sizes for each scenario, presenting a challenge in attempting to graph all
scenarios simultaneously due to these differing dimensions.

Reflection

This insight shed light on a significant aspect influencing our visualization approach. In
essence, this realization prompted us to reevaluate and fine-tune our strategy to accommodate
this variability effectively while ensuring the integrity and clarity of our results.

2nd Iteration

Design

The primary objective was to optimize computer memory usage without compromising data
integrity. We employed strategies to streamline storage while dynamically adapting to changing
matrix sizes, by creating n copies of a matrix while adjusting the row and column dimensions
accodingly.

Build

Incorporated into the Monte Carlo code, we developed an algorithm to address this challenge.
The algorithm compared matrices, identifying the required adjustments for each one without the
need for data discarding. When discrepancies in sizes were detected, the algorithm seamlessly
utilized the last obtained data from each scenario and repeated it as necessary until all
matrices were equal in size.

Test

After implementing the algorithm, we were able to successfully run the code and achieve our
primary objective of plotting each scenario. Nevertheless, upon comparison, the simulation
results did not align cohesively with the experimental results, which prompted an examination
of the model´s parameters.

Learn

The unexpected and unusual behaviors observed in the system led us to a critical realization -
certain parameters were inadequate. We couldn't find these parameters in the literature, and
upon reviewing our approach, we identified that the established ranges for the equations
constants were not sufficiently robust. This insight caused us to reevaluate and refine them
to ensure accuracy and reliability in our model, so our instructor, Ph.D. Segio Medina,
advised for parameter estimation.

Reflection

We emphasize the iterative nature of the DBTL cycle and the importance of adaptability.
Uncovered complexities and a disconnect between simulation and experimental results, reveals
the crucial role of accurate parameters in modeling. Though, we only had qualitative data of
the SDS-PAGE gel electrophoresis images, depicting inductions at different concentrations and
varying times for each sample.

3rd Iteration

Design

To analyze protein distribution comprehensively, we integrated quasi-quatitative data
extracted from ImageJ, an image analysis program. This data was coupled with a heuristic
algorithm designed to estimate the unknown kinetic parameters.

Build

The process consisted of defining protein bands and extracting concentration data across the
different gel lanes. Each gel lane corresponded to the same initial concentration, differing
only in terms of induction time points within the same sample. Additionally, assumptions were
established to streamline the calculation of endolysin concentration from total protein. The
data analyzed formed the basis for creating an objective function in a Genetic Algorithm (GA)
to calculate and minimize the error between experimental results and our model. The GA
iteratively optimized kinetic parameters of the ODEs system by comparing simulated protein
concentrations with experimental data.

Test

Utilizing the ODE15 solver in MATLAB to conduct the simulation based on the established model.
The simulation was run for each initial IPTG concentration. The primary evaluation criterion
was the convergence of the algorithm to a stable point, indicating that the GA effectively
minimized the error between simulated protein concentrations and the experimental data.
Another critical measure of success was the consistency in the converged point across several
generations of iterations

Learn

We observed consistent patterns in the graphs generated during the simulation, consistently
displaying three lines corresponding to the different induction concentrations. Notably, each
graph consistently stabilized at a non-zero point, suggesting that our solution space might be
overly complex or potentially suffering from overfitting. However, this was not a major
concern as the coefficients effectively described protein production under varying conditions.

Reflection

Across some of the graphs, several generations of iterations appeared as though the
optimization did not show apparent improvement. This observation hinted at the possibility
that the coordinates leading to the optimum for each line, specifically the minimum value,
remained consistent. The convergence to the same point in parameter estimation suggested a
stable and optimal solution for each line, indicating a robust and reliable estimation
process.

### Second sub-cycle: Protein Induction Kinetics

The next step in our process was to standardize the optimal conditions to induce our
fusion-proteins.

Click on each
accordion to see details

1st Iteration

Design

We planned to perform the protein synthesis by inducing our transformed *E. coli*
BL21(DE3) strains which contain the three plasmid constructions previously identified. For
this to be possible, we incorporated the biobrick BBa\_J435350 that codifies for the T7
promoter and also contains LacO regulations, inducible by the addition of IPTG. Our proteins
have a molecular weight of: 28.437 kDa, 35.098 kDa, and 24.359 kDa, for LysK-ABD-SH3B30,
LysCSA13-ABD, and PCNP-CecA-LysSS, respectively.

Build

The experiment was performed as described on our Experiments Page. Through this protocol, we
induced each endolysin once the inoculums reached 0.4–0.5 OD (600 nm) at 0.2 mM, 0.5 mM, and 1
mM IPTG. Two controls were added: the *E. coli BL21(DE3)* non-modified, and the *E.
coli BL21(DE3)* transformed non-induced. All of them were incubated for 6 hours. To
determine the optimal IPTG concentration to reach the highest level of protein production,
each hour's samples were harvested and lysed for their posterior SDS-PAGE analysis. In the
same way, we separated the soluble and insoluble fractions by centrifugation. Taking into
account the proteins’ weight, our polyacrylamide gels were prepared at 6% and 12% for the
concentrator and separator phases, respectively.

Test

Our results didn't show the LysCSA13-ABD (Figure 11) and the PCNP-CecA-LysSS(Figure 12)
proteins , but the LysK appeared successfully (Figure 13), and the protein soluble fraction
was identified (Figure 14).
![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/lyss.jpg)

**Figure 11.** LysCSA13-ABD 0.5 mM IPTG protein induction kinetic, 3th
hour. A) ColorBurst protein ladder. B) *E. coli BL21 (DE3)*-LysCSA13 non-inducted, zero
hour. C) *E. coli BL21 (DE3)*- LysCSA13 inducted at 0.5 mM, zero hour. D)*E. coli
BL21 (DE3)*-LysCSA13 non-inducted, first hour. E) *E. coli BL21 (DE3)*- LysCSA13
inducted at 0.5 mM, first hour. F)*E. coli BL21 (DE3)*-LysCSA13 non-inducted, second
hour. G) *E. coli BL21 (DE3)*- LysCSA13 inducted at 0.5 mM, second hour. H) *E. coli
BL21 (DE3)*-LysCSA13 non-inducted, third hour. I) *E. coli BL21 (DE3)*- LysCSA13
inducted at 0.5 mM, third hour.

![](https://static.igem.wiki/teams/4607/wiki/silver/engineering-lab/lysssreal.jpg)

**Figure 12.** PCNP-CecA-LysSS 0.2 mM IPTG protein induction kinetic, 4th
to 6th hour. A) Precision Plus Protein Dual Color Standards (10–250 kD) ladder. B) *E. coli
BL21 (DE3)*. C) *E. coli BL21 (DE3)*- PCNP-CecA-LysSS non-inducted, fourth hour.
D) *E. coli BL21 (DE3)*- PCNP-CecA-LysSS inducted at 0.2 mM IPTG, fourth hour. E)
*E. coli BL21 (DE3)*- PCNP-CecA-LysSS non-inducted, fifth hour. F) *E. coli BL21
(DE3)*- PCNP-CecA-LysSS inducted at 0.2 mM IPTG, fifth hour. G) *E. coli BL21
(DE3)*- PCNP-CecA-LysSS non-inducted, sixth hour. H) *E. coli BL21 (DE3)*-
PCNP-CecA-LysSS inducted at 0.2 mM IPTG, sixth hour. I) *E. coli BL21 (DE3)*, first
hour.

![](
                          https://static.igem.wiki/teams/4607/wiki/parts/sds/lysk-abd-sh3b30-kinetic-sds.jpg
                          )

**Figure 13.** LysK-ABD-SH3B30 0.2 mM IPTG protein induction kinetic, 4th
to 6th hour. A) ColorBurst protein ladder. B) *E. coli BL21 (DE3)*. C) *E. coli BL21
(DE3)*- LysK-ABD-SH3B30 non-inducted, fourth hour. D) *E. coli BL21 (DE3)*-
LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, fourth hour. E) *E. coli BL21 (DE3)*-
LysK-ABD-SH3B30 non-inducted, fifth hour. F) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30
inducted at 0.2 mM IPTG, fifth hour. G) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30
non-inducted, sixth hour. H) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30 inducted at 0.2 mM
IPTG, sixth hour. I) *E. coli BL21 (DE3)*, first hour.

![](
                        https://static.igem.wiki/teams/4607/wiki/parts/sds/sds-fracciones-lysk-2.jpg
                        )

**Figure 14.** LysK-ABD-SH3B30 soluble (A) and insoluble (B) fractions. Visualization
through a polyacrilamide gel 12% (SDS-PAGE) results. Both gels present the same distribution
of wells, each one corresponding to the fraction (soluble or insoluble) assigned.

 A)
MWM (for gel A: Precision Plus Protein Dual Color Standards (10–250 kD), for gel B: PageRuler™
Plus Prestained Protein Ladder, 10 to 250 kDa). B) *E. coli BL21 (DE3)* C) *E. coli
BL21 (DE3)*- LysK-ABD-SH3B30 non-inducted. D) *E. coli BL21 (DE3)*-
LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, 2 μL. E) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30
inducted at 0.2 mM IPTG, 4 μL. F) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30 inducted at 0.2
mM IPTG, 6 μL. G) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, 8 μL.
H) *E. coli BL21 (DE3)*- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, 10 μL. I) *E. coli
BL21 (DE3)*- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, 15 μL. J) *E. coli BL21
(DE3)*- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, 20 μL.

Learn

As shown in the test stage, the experimentation revealed that some of the samples didn't
appear in the SDS-PAGE, while others did. All the processes were prepared and performed in the
same way. In the first place, we could see that, for LysK-ABD-SH3B30 the proper bands
appeared, in which we could identify that the best IPTG concentration was 0.2 mM for 5 hours
and the protein was in the soluble fraction. Roughly speaking, we confirmed that our
construction was correctly designed. On the other hand, LysCSA13-ABD didn’t show bands for any
of the samples, but we know the gel was correctly run because of the protein ladder
appearance. In addition, the PCNP-CecA-LysSS only showed results in the negative controls
(non-induced, and non-transformed strains).

Reflection

While it is true that the results for this iteration included good news in the development of
the product, the obstacles were really challenging. To analyze the situation, we actually know
that all the processes were the same for each of the samples, but the results were completely
different for each one. Knowing that LysK-ABD-SH3B30 achieved the expected results, we can be
assured that our protocols work correctly. Taking into account that for LysCSA13-ABD none of
the samples showed bands, we assumed that the problem was on the lysis step since we are
completely sure that the cell growth was the same for each of the samples, as was verified
through the optical density. In that case, our first question for the second iteration was:
"Was the lysis exactly the same for each sample? How can we improve the lysis efficiency?"
Considering that the process depends completely on temperature, we know how to counteract this
kind of issue. To solve this problem, we needed to prolong the incubation time.
In the case of the PCNP-CecA-LysSS, the challenge looked harder. Evaluating that only the
transformed strains showed no bands, we were completely confident that something about our
protein was affecting cell growth, but it also confirmed that our construction worked. Our
main hypothesis was that our protein, which lyses *E. coli* strains, was currently
toxic for our host. In the background research, we didn’t find anything about killing the
specific *E. coli BL21(DE3)* strain by expressing each of the parts that compound our
fusion-endolysin, but results demonstrated that it could be occurring. The main question to
solve is: "Was our protein lysing the bacteria? What can we do to prevent it?" To be assured
that our results were right, we planned to repeat the experiment before assuming that our
hypothesis could be true.

2nd Iteration

Design

Before evaluating the first iteration of this semi-cycle, we decided to check our hypothesis
by changing the lysis incubation. Considering that the LysK protein revealed that the process
worked, and as a consequence of researching other ways to lysate without using additional
reagents, we concluded that extending the incubation time from 5 min up to 10 min and adding a
posterior step in boiling water for 5 min, there was no way of having unlysed cells in the
SDS-PAGE wells.

Build

We performed the protocol for the protein induction kinetics as described in our Experiments
Page, taking into account the lysis incubation changes. We induced both endolysins once the
inoculums reached 0.4–0.5 OD (600 nm) at 0.2 mM, 0.5 mM, and 1 mM IPTG. Two controls were
added: the *E. coli BL21(DE3)* non-modified, and the *E. coli BL21(DE3)*
transformed non-induced. All of them were incubated for 6 hours. To determine the optimal IPTG
concentration to reach the highest level of protein production, each hour's samples were
harvested and heat-lysed for their posterior SDS-PAGE analysis. In the same way, we separated
the soluble and insoluble fractions by centrifugation. Taking into account the proteins’
weight, our polyacrylamide gels were prepared at 6% and 12% for the concentrator and separator
phases, respectively.

Test

After the induction and SDS-PAGE electrophoresis we identified the expected LysCSA13-ABD
bands, as well as its optimal conditions at 0.2 mM for 6 hours (Figure 15). In addition, we
identified that this protein was in the insoluble fraction (Figure 16). On the other hand,
PCNP-CecA-LysSS notice better bands, but only for the negative controls.
![](https://static.igem.wiki/teams/4607/wiki/parts/lyscsa13-abd-kinetic-sds.jpg)

*Figure 15* LysCSA13-ABD 0.2 mM IPTG protein induction kinetic. A) ColorBurst protein
ladder. B) *E. coli BL21 (DE3)*. C) *E. coli BL21 (DE3)*- LysCSA13-ABD
non-inducted, fourth hour. D) *E. coli BL21 (DE3)*- LysCSA13-ABD inducted at 0.2 mM
IPTG, fourth hour. E) *E. coli BL21 (DE3)*- LysCSA13-ABD non-inducted, fifth hour. F)
*E. coli BL21 (DE3)*- LysCSA13-ABD inducted at 0.2 mM IPTG, fifth hour. G) *E. coli
BL21 (DE3)*- LysCSA13-ABD non-inducted, sixth hour. H) *E. coli BL21 (DE3)*-
LysCSA13-ABD inducted at 0.2 mM IPTG, sixth hour. I) *E. coli BL21 (DE3)*, first hour.

![](https://static.igem.wiki/teams/4607/wiki/parts/900-px-lyscsa13-abb-insol-and-sol.jpg)

**Figure 5.** LysCSA13-ABD soluble (A) and insoluble (B) fractions. Visualization through
a polyacrilamide gel 12% (SDS-PAGE) results. Both gels present the same distribution of wells,
each one corresponding to the fraction (soluble or insoluble) assigned. A) MWM (for gel A:
Precision Plus Protein Dual Color Standards (10–250 kD), for gel B: PageRuler™ Plus Prestained
Protein Ladder, 10 to 250 kDa). B) *E. coli BL21 (DE3)*. C) *E. coli BL21
(DE3)*- LysCSA13-ABD non-inducted. D) *E. coli BL21 (DE3)*- LysCSA13-ABD inducted
at 0.2 mM IPTG, 2 μL. E) *E. coli BL21 (DE3)*- LysCSA13-ABD inducted at 0.2 mM IPTG, 4
μL. F) *E. coli BL21 (DE3)*- LysCSA13-ABD inducted at 0.2 mM IPTG, 6 μL. G) *E. coli
BL21 (DE3)*- LysCSA13-ABD inducted at 0.2 mM IPTG, 8 μL. H) *E. coli BL21 (DE3)*-
LysCSA13-ABD inducted at 0.2 mM IPTG, 10 μL. I) *E. coli BL21 (DE3)*- LysCSA13-ABD
inducted at 0.2 mM IPTG, 15 μL. J) *E. coli BL21 (DE3)*- LysCSA13-ABD inducted at 0.2
mM IPTG, 20 μL. K)

Learn

As shown in the test stage, increasing the incubation time and incorporating a posterior phase
in boiling water improved the lysis method. For this iteration, the protein LysCSA13-ABD
appeared and matched the expected band weight. We identified that the optimal conditions were
induction at 0.2 mM IPTG with an incubation period of 6 hours. In the same way, we confirmed
that it’s an insoluble protein. Unfortunately, the modification only improved the lysis phase,
and for the PCNP-CecA-LysSS protein, the problem still appears.

Reflection

By using the engineering cycle, one of our current problems was solved. In the same way, we
verified that the PCNP-CecA-LysSS issues were not related to the lysis phase. Our hypothesis
about the toxicity of our protein was more accurate. In fact, it confirms that our protein was
correctly designed and possesses functional enzymatic activity. The new question was about how
we could prevent the early death of our cell host and effectively produce our third endolysin.

3rd Iteration

Design

After reflecting on our drawbacks, we realized that our PCNP-CecA-LysSS protein lyses the cell host because of the differences on the culture turbidity between the inducted samples and the non-inducted ones. To identify that our hypothesis was right we considered running an SDS electrophoresis, in that case, we evaluated the possibility of increasing the acrylamide concentration up to 15% and 8% for the separator and concentrator phases respectively. In the same way, we planned to take a sample from our final crude protein extract, to be assured of the presence of our protein.

Build

We performed massive protein inductions at 0.2 mM, 0.5 mM, and 1.0 mM IPTG concentrations of our PCNP-CecA-LysSS at 37°C for 6 hours, once our inoculums reach 0.4 - 0.6 OD (600 nm). We also included BL21 and non-inducted negative controls. To lyse our inducted cells, we use the B-PER reagent as is described in the Experiments page. After the protein extraction, a SDS-PAGE at 15% and 8% polyacrylamide concentrations (separator and concentrator phases).

Test

We performed an SDS-PAGE to identify the expected protein bands for the PCNP-CecA-LysSS, which has a molecular weight of 24 kDa. The results can be observed at Figure 17.
![](https://static.igem.wiki/teams/4607/wiki/parts/lysssreal2-0.jpg)

**Figure 17. PCNP-CecA-LysSS expression. Visualization through a polyacrilamide gel 15% (SDS-PAGE) results.**  *A) MWM (iBright). B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- PCNP-CecA-LysSS non-inducted. D) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 0.2 mM IPTG. E) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 0.5 mM IPTG. F) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 1.0 mM IPTG*

Learn

The protein expression of our third endolysin was identified. Considering the principles of catalytic activity of this endolysin, we realized that the toxicity of the host cell hinders adequate protein production, in this way we addressed the problem by increasing the final concentration of proteins. This was possible by analyzing the crude protein extracts at different concentrations of IPTG.

Reflection

Reflecting on the Engineering cycle gave us the opportunity to affront and resolve one of our biggest problems. In that case, we finally identified the presence of our protein in our IPTG inductions by increasing the final concentration of protein for the sample. Generally, we only took samples of 200 uL of bacterium, but for this case, 20 uL of the final crude protein of 20 mL was lysed and charged in our SDS-PAGE.

![](https://static.igem.wiki/teams/4607/wiki/entrepreneurship/entrepreneurship/home/img-1653-1.jpg)

# Team

# Experiments

Overview

Round zero

First round

Second round

Third round

Fourth round

Protocols

References

# Experimentation is the greatest science

Overview

![Experimental Design Diagram](https://static.igem.wiki/teams/4607/wiki/experiments/experimentaldes.png)

Our experimental design covers the set of
procedures
we executed in order to successfully
produce our recombinant endolysins. Covering
everything from our plasmid construction to the
bactericidal assays, this page explains our
journey
through the laboratory.

Round Zero: Plasmid
Construction and Analysis

![Round Zero: Plasmid Construction and Analysis Diagram](https://static.igem.wiki/teams/4607/wiki/experiments/zerorec.png)

The initial round of experimentation encompassed
the
*in silico*  design of our plasmids,
their synthesis, assembly, transformation, and
analysis of the constructs. This comprehensive
phase integrates computational design with
practical
implementation, culminating in the
evaluation of the resulting constructs.
To design our endolysins coding sequences, we
conducted an in-depth protein analysis. This
helped us to identify the crucial domains
required
to produce highly effective and catalytically
specific endolysins. The coding sequence (CDS)
of
the three recombinant intein-fusion proteins
(LysK
endolysin,
LysCSA13, and LysSS) was *in silico*
edited
to incorporate BsaI enzyme recognition sites
at the ends of the sequence. In the same way,
each
CDS design was evaluated through the
protein structural model analysis tool, Alpha
Fold
2, to identify the original functional
protein structures and compare them with our
final
designs, ensuring their enzymatic activity.
Afterwards, we proceeded with the selection of
the
bioparts to be
utilized in the assembly of the transcriptional
units (TUs) that would express our proteins.

The selected [parts](https://2023.igem.wiki/tec-chihuahua/parts)
for the construction of the transcriptional
units
were:

1. ### Promoter

   [BBa\_J435350](http://parts.igem.org/Part%3ABBa_J435350)
2. ### RBS

   [BBa\_Z0262](http://parts.igem.org/Part%3ABBa_Z0262)
3. ### Terminator

   [BBa\_J435371](http://parts.igem.org/Part%3ABBa_J435371)
4. ### Backbone

   [BBa\_J435330](http://parts.igem.org/Part%3ABBa_J435330)

These parts were assembled in
SnapGene® software using the Golden Gate
method,
employing the type IIS restriction enzyme
BsaI.The transcriptional units (TUs) of each
of
our
endolysins (TU LysK, TU LysCSA13, and TU
LysSS)
were synthesized by Twist Bioscience, making
this possible by the sponsorship of 10 kb of
FREE Twist Gene Fragments for iGEM teams.

First Round:
Golden Gate Assembly

![First Round: Golden Gate Assembly Diagram](https://static.igem.wiki/teams/4607/wiki/experiments/firstrec.png)

From the iGEM 2023 plate, the BioBrick™
BBa\_J435330
(plate 2, well A15) was [resuspended,
which was [transformed](https://static.igem.wiki/teams/4607/wiki/experiments/experiments/biobrick-dna-resuspension.pdf) into *E. coli*
5-alpha by heat shock, and subsequently
[extracted
and purified](https://static.igem.wiki/teams/4607/wiki//experiments/miniprep-reaction.pdf) with the PureLink™
Quick
Plasmid Miniprep Kit (Invitrogen™, Lot
#01260760). This part was chosen as the
backbone
due
to its high replication origin, dual
antibiotic
resistance (kanamycin and ampicillin), and
compatibility with the Golden Gate assembly
method. Once the BBa\_J435330 backbone was
extracted and purified, [Golden
Gate assembly reactions](https://static.igem.wiki/teams/4607/wiki//experiments/golden-gate-assembly.pdf) were
performed
using the BsaI
enzyme (NEB E1601, Lot #10182427) to insert
each
of the transcriptional units into the
BBa\_J435330 backbone.

After the Golden Gate assembly reaction, a
bacterial [transformation](https://static.igem.wiki/teams/4607/wiki//experiments/heat-shock-cell-s-transformation.pdf)
into *E. coli* 5-alpha was performed,
followed by
plating on LB agar with kanamycin (50
µg/mL).
However, colonies were obtained both on
the plates seeded with the transformation
and in
the negative controls. This led us to
suspect that the plates from the Golden Gate
reaction might contain a mixture of
colonies transformed with the target
construct
and others transformed with the backbone
alone, making them indistinguishable by
visual
inspection.

To confirm this, re-plating of several
colonies
from the same plate was conducted onto
other plates with kanamycin and ampicillin.
This
was done in order to take advantage of
the dual antibiotic resistance feature
present
in the used backbone. A colony from the
reaction-seeded plate was selected and then
plated onto two separate plates: one with
kanamycin (50 µg/mL) and another with
ampicillin
(100 µg/mL). The same identifying
number was assigned to both antibiotics.
The next day, observation revealed that some
colonies grew on both antibiotics, while
specific colonies only grew on kanamycin (50
µg/mL) and on ampicillin there wasn't any
growth (100 µg/mL). This suggested that
these
colonies might have been transformed with
a construct carrying resistance only to
kanamycin (50 µg/mL) and not to ampicillin.
This
led us to suspect that our construct might
indeed be present in these colonies.

To verify this, a colony that exhibited only
kanamycin resistance (50 µg/mL) was
selected. DNA plasmid extraction and
purification were performed using the
PureLink™
Quick Plasmid Miniprep Kit (Invitrogen™, Lot
#01260760).
With the aim of assessing the effectiveness
of
the Golden Gate assembly protocol, we
conducted an enzymatic digestion to
linearize
the plasmids, followed by an analysis
through agarose gel electrophoresis. We cut
pUC-LysK and pUC-LysCSA13 to digestion using
the SmaI enzyme, and pUC-LysSS underwent
digestion using the EcoRI enzyme.](https://static.igem.wiki/teams/4607/wiki/experiments/experiments/biobrick-dna-resuspension.pdf)

Second Round:
Protein Expression

![Second Round: Protein Expression Diagram](https://static.igem.wiki/teams/4607/wiki/experiments/secondrec.png)

The next step of our recombinant protein
production process was grouped into the
second
round of
experimentation. All the selection,
optimization, and standardization of our
protein
induction
protocols were performed as follows:

**### Protein Production**

Our constructs incorporate the biobrick [BBa\_J435350](http://parts.igem.org/Part%3ABBa_J435350),
featuring a T7 promoter with LacO regulatory
regions. This choice was driven by its
capability for high transcription
levels and effective regulation. To induce
protein production in our *E. coli*
BL21
(DE3) transformants carrying
pUC-LysCSA13 & pUC-LysK, we employed
isopropyl
β-D-1 thiogalactopyranoside (IPTG) as an
inducer
for the LacO regulation
regions. This reagent activates the
transformants, enabling them to synthesize
our
endolysins.

**### Protein Induction Kinetics**

To determine the optimal protein induction
parameters, we conducted
[protein
induction kinetics](https://static.igem.wiki/teams/4607/wiki//experiments/iptg-protein-induction-kinetic.pdf). Following prior
research, we established that
the ideal conditions were approximately 6
hours
at 37°C. Experiments were conducted at three
IPTG concentrations—0.2
mM, 0.5 mM, and 1 mM—across all
transformants
using *E. coli* BL21 (DE3) strains
transformed with plasmids
obtained via Golden Gate Assembly.

Protein induction kinetics were carried out
at
37°C for a maximum of 6 hours with vigorous
agitation in an orbital
shaker. Each endolysin underwent five
treatments: a negative control with *E.
coli*
BL21 (DE3), a negative control
with *E. coli* BL21 (DE3) transformed
with the endolysin but without IPTG
induction,
and three *E. coli*
BL21 (DE3) transformed with the endolysin
induced at different IPTG concentrations—0.2
mM,
0.5 mM, and 1 mM. Samples were
collected hourly, yielding 200 μL for each
treatment.
Next, we performed SDS-PAGE analysis. Given
that
the molecular weight of LysCSA13-ABD is
35.098
kDa, and LysK-ABD-SH3B30
weighs 28.437 kDa, we used 12% and 6%
concentrations for the separator and
concentrator phases, respectively.

**### Fraction Identification**

Finally, once the optimal concentration and
induction time parameters were established
for
LysK and LysCSA13,
an SDS-PAGE was performed using the soluble
and
insoluble fractions from each of the crude
bacterial lysis
extracts after induction. This step was
crucial
in assessing the solubility of both
endolysins
and, consequently,
ensuring accurate purification.

Third Round:
Protein Purification

![Third Round: Protein Purification Diagram](https://static.igem.wiki/teams/4607/wiki/experiments/thirdrec.png)

After phase two, valuable information was
gained
regarding LysCSA13-ABD and LysK-ABD-SH3B30.
Specifically, it enabled us to determine the
optimal induction conditions and solubility
of
each.
This paved the way for the subsequent round
of
experimentation: purification through metal
affinity
chromatography for His-tagged proteins
(Thermo
Scientific™, 88228).

**### Protein Purification**

For this phase, we conducted a [massive
induction](https://static.igem.wiki/teams/4607/wiki//experiments/iptg-protein-induction-kinetic.pdf) to produce our
endolysins in greater quantities.
Subsequently,
we proceeded with purification through
metal affinity chromatography using
centrifugation columns.

**### Validation of the Endolysin Purification Process**

In order to confirm the successful isolation
of
our recombinant proteins through the
purification process, an analysis was
conducted
using SDS-PAGE. This involved verifying
the eluate from the purification and
contrasting
it with two negative controls:
non-transformed
*E. coli* and non-induced transformed
*E.
coli*. Additionally, the molecular
weights
of the bands obtained in the polyacrylamide
gel
were confirmed.

Fourth Round:
Bactericidal Assays

![Fourth Round: Bactericidal Assays Diagram](https://static.igem.wiki/teams/4607/wiki/experiments/fourthrec.png)

For this stage the main purpose was to corroborate the catalytic activity of our endolysins, the optimal parameters, and the real effects for each bacteria. In the same way, the chitosan-alginate delivery drug system was analyzed simultaneously with our enzymatic treatment.

Considering our protein purification results, we decided to work on the development of our treatment by using our crude protein extracts. To identify our optimal composition, we designed an experimental model which makes possible the enzyme simultaneous analysis and its effects on the pathogenic bacteria. The full protocol is described in our [Experiments Page](https://2023.igem.wiki/tec-chihuahua/experiments). Our ELISA plate designing was prepared as follows:

![](https://static.igem.wiki/teams/4607/wiki/plate1-enzymes.jpg)

**Figure 1. Distribution of ELISA plates for bactericidal assays.**

According to the previous figure, all the raw endolysin extract wells contains not only the enzyme combination, but also 90 uL from the pathogenic bacterium desired. In our design, a range of 3 rows is requiered for each repetition, and at least 3 are required for the experimental validation (For each bacteria). To evaluate our results, we decided to apply the Response Surface Methodology, which is deeply described in our [Model Page](https://2023.igem.wiki/tec-chihuahua/model).

 For the validation of our protein-loaded chitosan alginate beads the experimental design was prepared as follows:

![](https://static.igem.wiki/teams/4607/wiki/placaquitosan.jpg)

**Figure 2. Distribution of ELISA plates for bactericidal assays with loaded protein chitosan alginate beads.**

Protocols

[Biobrick DNA
Resuspension](https://static.igem.wiki/teams/4607/wiki/experiments/experiments/biobrick-dna-resuspension.pdf)

[*E. coli*
Chemically
Competent Cells](https://static.igem.wiki/teams/4607/wiki/experiments/competent.pdf)

[Heat Shock Cell’s
Transformation](https://static.igem.wiki/teams/4607/wiki//experiments/heat-shock-cell-s-transformation.pdf)

[Miniprep](https://static.igem.wiki/teams/4607/wiki//experiments/miniprep-reaction.pdf)

[Golden Gate Assembly](https://static.igem.wiki/teams/4607/wiki//experiments/golden-gate-assembly.pdf)

[Agarose Electrophoresis](https://static.igem.wiki/teams/4607/wiki//experiments/agarose-electrophoresis.pdf)

[Nanodrop 2000 DNA
Quantification](https://static.igem.wiki/teams/4607/wiki//experiments/nanodrop-2000-dna-quantification.pdf)

[IPTG Protein Induction
Kinetic](https://static.igem.wiki/teams/4607/wiki//experiments/iptg-protein-induction-kinetic.pdf)

[Massive IPTG Protein
Induction](https://static.igem.wiki/teams/4607/wiki//experiments/massive-iptg-protein-induction.pdf)

[B-Per Bacterial Protein
Extraction](https://static.igem.wiki/teams/4607/wiki//experiments/b-per-bacterial-protein-extraction.pdf)

[SDS-PAGE](https://static.igem.wiki/teams/4607/wiki//experiments/sds-page.pdf)

[HisPur™ Ni-NTA Protein
Purification](https://static.igem.wiki/teams/4607/wiki//experiments/hispur-ni-nta-protein-purification.pdf)

[Protein Quantification
on
NanoDrop 2000](https://static.igem.wiki/teams/4607/wiki/experiments/proteinquan.pdf)

[Antimicrobial Assays](https://static.igem.wiki/teams/4607/wiki/experiments/bactericidal-assays.pdf)

[Solution Preparation](https://static.igem.wiki/teams/4607/wiki//experiments/protocolos-anexos.pdf)

[Full Notebook](https://static.igem.wiki/teams/4607/wiki/experiments/labnotebook-tec-chihuahua.pdf)

References

(1) Addgene (2016). Addgene: Pouring LB Agar Plates. [online] Addgene.org. Available at: https://www.addgene.org/protocols/pouring-lb-agar-plates/.

(2) Cha, Y., Son, B., & Ryu, S. (2019). Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. Food Microbiology, 103245. doi:10.1016/j.fm.2019.103245

(3) New England Biolabs. (2019). High Efficiency Transformation Protocol (C2987H/C2987I) | NEB. Neb.com. https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987

(4) iGEM (2023). iGEM Technology. [online] technology.igem.org. Available at: https://technology.igem.org/distribution/handbook [Accessed 24 Jun. 2023].

(5) Seaver, S. (2014). How to make a 25-50 mg/ml Chloramphenicol Stock Solution v1. doi:https://doi.org/10.17504/protocols.io.ci5ug5.

(6) Sanz-Gaitero, M., Keary, R., Garcia-Doval, C., Coffey, A., & van Raaij, M. J. (2013). Crystallization of the CHAP domain of the endolysin from Staphylococcus aureus bacteriophage K. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications, 69(Pt 12), 1393–1396. https://doi.org/10.1107/S1744309113030133

(7) Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved biodistribution and extended serum half-life of a bacteriophage endolysin by albumin binding domain fusion. Frontiers in Microbiology, 9, 2927. https://doi.org/10.3389/fmicb.2018.02927

![](https://static.igem.wiki/teams/4607/wiki/project-description/whatsapp-image-2023-08-27-at-15-34-55.jpeg)

# Team

# Results

Overview

Achievements

Round Zero

First Round

Second Round

Third Round

Fourth Round

References

# "There is no science without results"

## Overview

AureoBos's general purpose is to develop a novel solution for
bovine mastitis by approaching it from an
unexplored side in Mexico: synthetic biology. Hence, our project's
path starts in functional BioBricks. By
analyzing various molecular mechanisms existing in nature,
endolysins caught our attention because of
their mechanism and specificity.
By mimicking the endolysin's mechanism, our lab developed
antimicrobial fusion proteins that fight four of
the main microorganisms known to cause bovine mastitis: *Staphylococcus
aureus*, *Streptococcus uberis*,
*Streptococcus agalactiae*. and *Escherichia coli*.
Throughout this page you can find the
bioparts used and designed to construct the expression cassettes
that paved the way of this project.

## Achievements

## 00

### Round Zero

**Succesful registry of eight new basic parts, and three
new composite parts
that were modified by Golden Gate assembly.**

[Read
More](#sec3)

## 01

### First Round

**Successful assembly of transcriptional units in the
acceptor vector via Golden Gate assembly.**

[Read
More](#sec4)

## 02

### Second Round

**Optimal induction conditions and solubility of our
endolysins identified.**

[Read
More](#sec5)

## 03

### Third Round

**Successful purification of endolysin LysK through
metal affinity chromatography in spin columns.**

[Read
More](#sec6)

## 04

### Fourth Round

**Bactericidal Assays**

[Read
More](#sec7)

## Round Zero: Plasmid Construction and Analysis

The initial round of experimentation encompassed the *in silico*
design of our plasmids, their synthesis,
assembly, transformation, and analysis of the constructs. This
comprehensive phase integrates
computational design with practical implementation, culminating in
the evaluation of the resulting
constructs.
To design our endolysins coding sequences, we conducted an
in-depth
protein analysis. This helped us to
identify the crucial domains required to produce highly effective
and catalytically specific endolysins.
The resulting sequences encode the following intein-fusion
proteins:

### Composite Parts

Expression cassette for LysCSA13-ABD protein

**Fusion endolysin against *S. aureus*
bacteria
who causes bovine mastitis.**

![LysCSA13-ABD protein](https://static.igem.wiki/teams/4607/wiki/parts/dn/lyscsa13-abd.png)

***Figure 1.*** LysCSA13-ABD
protein diagram.

This part contains the linear sequence of the
LysCSA13-ABD
([BBa\_K4607000](http://parts.igem.org/Part%3ABBa_K4607000))
nucleotide optimized for *E.
coli*. It incorporates some of the most
efficient
biobricks as described below: the T7
promoter with LacO regulations BBa\_J435350, the medium
strength RBS BBa\_Z0262, and the triple
terminator BBa\_J435371. For our plasmid construction,
we
used the high copy pUC ori /Kan R backbone
BBa\_J435330. It also contains the BBa\_K4607000 that
codifies for a fusion protein based on the
endolysin Lys from Staphylococcus aureus virulent
bacteriophage CSA13, which have the ability to
lyse *S. aureus* with very specific targeting and an
excellent catalytic activity.[1,2,3](#referencia1)

Expression cassette for LysK-ABD-SH3B30 protein

**Fusion endolysin against *S. aureus*
bacteria
who causes bovine mastitis.**

![LysCSA13-ABD protein](https://static.igem.wiki/teams/4607/wiki/parts/dn/lysk-abd.png)

***Figure 2.*** LysCSA13-ABD
protein diagram.

This part contains the linear sequence of the
LysK-ABD-SH3B30 ([BBa\_K4607001](http://parts.igem.org/Part%3ABBa_K4607001))
nucleotide optimized for *E.
coli*. It incorporates some of the most efficient
biobricks as described below: the T7
promoter with LacO regulations BBa\_J435350, the medium
strength RBS BBa\_Z0262, the triple terminator
BBa\_J435371, and the high copy pUC ori/Kan R backbone
BBa\_J435330. It also contains the BBa\_K4607001
that codifies for a fusion of the CHAP domain from the
Lys
of the bacteriophage K, with the ability
to degrade the cell wall of antibiotic-resistant strains
of *Staphylococcus aureus*, as well
as, *S. agalactiae* and *S. uberis*.[4,5,6,2](#referencia2)

Expression cassette for PCNP-CecA-LysSS protein

**Fusion artilysin against *E. coli***

![PCNP-CecA-LysSS](https://static.igem.wiki/teams/4607/wiki/parts/dn/pcnp-ceca-lysss.png)

***Figure 3.*** PCNP-CecA-LysSS
expression cassette diagram.

This part contains the linear sequence of the
PCNP-CecA-LysSS nucleotide optimized for *E.
coli*. It incorporates some of the most efficient
biobricks as described below: the T7
promoter with LacO regulation, the medium strength RBS,
and the triple terminator. For our plasmid
construction, we used the high copy pUC ori /Kan R
backbone. It also contains a fusion protein based
on three main parts: the polycationic nonapeptide
(PCNP),
Cecropin A peptide, and the LysSS protein,
all integrated with flexible linkers that assure its
functionality.[7,8,9](#referencia7)

The coding sequence (CDS) of the three recombinant intein-fusion
proteins (LysK endolysin, LysCSA13, and
LysSS)
was  *in silico* edited to incorporate BsaI enzyme
recognition sites at the ends of the sequence.
In
the same way, each CDS design has been evaluated through the
protein
structural model analysis tool, Alpha
Fold 2, to identify the original functional protein structures and
compare them with our final designs,
ensuring their enzymatic activity (Figure 4 - 6).A great quantity of protein chains can be predicted accurately by using the AlphaFold 1 model; this model has the possibility to predict protein structures from a dataset of 4446 protein complexes, without non-redundant interfaces. The AlphaFold 2.0 offers a very high accuracy in the Critical Assessment of Protein Structure Prediction, and by the 2021 the AlphaFold Protein Structure Database reach an unprecedented group of reliability completely accessible to the scientific community. Thanks to its algorithm it is possible to simulate three-dimensional models for the whole human proteome, and a high variety of proteins.1–3 The best
LysCSA13-ABD option was developed by
identifying
that the presence of 6x HisTag (c) affects the functional protein
domains (Figure 4) in comparison to the
original LysCSA13 (a) and LysCSA13-ABD (b) structures. To solve
this
problem, we added a TEV site to its
posterior removal (d). In the case of LysK-ABD-SH3B30 (Figure 5),
we
realized that the addition of the X6
HisTag (c) didn’t affect the protein’s functionality, and in
comparison between the original LysK (a) and
LysK-ABD-SH3B0 (d) sequences, no issue was encountered. Finally,
(Figure 6) LysSS demonstrated that using
short flexible linkers[4,5](#referencia4), any of the original peptide sequences (a),
(b), or LysSS original protein sequence
(c) would have been affected, so the final design (d) conserved
its
enzymatic activity. Additionally, we
assured that all our final intein-fusion proteins kept the
original
proteins’ distribution with functional
domains.[6-9](#referencia6)

![](https://static.igem.wiki/teams/4607/wiki/parts/todas-las-proteinas-de-lyscsa13-alphafold2-v2.png)

**Figure 4.** Alpha Fold 2’s
LysCSA13-ABD protein
folding
analysis.
(a) Original folding from LysCSA13 . (b) Folding of LysCSA13-ABD
(c)
LysCSA13-ABD-6X HisTag’s folding. (d) LysCSA13-ABD-TEV Site-X6
HisTag’s folding.

![](https://static.igem.wiki/teams/4607/wiki/parts/todas-las-proteinas-de-lysk-alpha-fold2-v2.png)

**Figure 5.** Alpha Fold 2’s
LysK-ABD-SH3B30
protein folding
analysis.
(a) Original folding from LysK . (b) LysK-ABD-SH3B30-TEV Site-6X
HisTag’s
folding (c) LysK-ABD-SH3B30-X6 HisTag’s folding. (d)
LysK-ABD-SH3B30’s folding.

![](https://static.igem.wiki/teams/4607/wiki/parts/todas-las-proteinas-de-lysss-en-alpha-fold2-v2.png)

**Figure 6.** Alpha Fold 2’s
PCNP-CecA-LysSS
protein folding
analysis.
(a) Folding from the PCNP fused with OBPgp279 protein. (b)
CecA-Lys10-24
protein’s folding (c) Original LysSS’s folding. (d)
PCNP-CecA-LysSS-6X HisTag’s folding.

Parts were assembled in SnapGene® software using the Golden
Gate
method, employing the type IIS
restriction enzyme BsaI.
The transcriptional units (TUs) of each of our endolysins (TU
LysK,
TU LysCSA13, and TU LysSS) were
synthesized by Twist Bioscience, making this possible by the
sponsorship of 10 kb of FREE Twist Gene
Fragments for iGEM teams. This genetic material was delivered
lyophilized with a total of 1000 ng per
tube, which were resuspended in 100 µL of TE to achieve a final
concentration of 10 ng/mL for each of the
transcriptional units. Subsequently, aliquots of each TU were
prepared to proceed with the Golden Gate
assembly reaction.

## First Round: Golden Gate Assembly

### Golden Gate Assembly Reaction

From the iGEM 2023 plate, the BioBrick™ BBa\_J435330 (plate 2, well
A15) was resuspended, which was
transformed into *E. coli* 5-alpha by heat shock, and
subsequently extracted and purified with the
PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760).
This part was chosen as the backbone
due to its high replication origin, dual antibiotic resistance
(kanamycin and ampicillin), and
compatibility with the Golden Gate assembly method. Once the
BBa\_J435330 backbone was extracted and
purified, Golden Gate assembly reactions were performed using the
BsaI enzyme (NEB E1601, Lot #10182427)
to insert each of the transcriptional units into the BBa\_J435330
backbone.

After the Golden Gate assembly reaction, a bacterial
transformation
into *E. coli* 5-alpha was
performed, followed by plating on LB agar with kanamycin (50
µg/mL).
However, colonies were obtained both
on the plates seeded with the transformation and in the negative
controls. This led us to suspect that the
plates from the Golden Gate reaction might contain a mixture of
colonies transformed with the target
construct and others transformed with the backbone alone, making
them indistinguishable by visual
inspection.

To confirm this, re-plating of several colonies from the same
plate
was conducted onto other plates with
kanamycin and ampicillin. This was done in order to take advantage
of the dual antibiotic resistance
feature present in the used backbone. A colony from the
reaction-seeded plate was selected and then plated
onto two separate plates: one with kanamycin (50 µg/mL) and
another
with ampicillin (100 µg/mL). The same
identifying number was assigned to both antibiotics. The next day,
observation revealed that some colonies
grew on both antibiotics, while specific colonies only grew on
kanamycin (50 µg/mL) and on ampicillin
there wasn't any growth (100 µg/mL). This suggested that these
colonies might have been transformed with a
construct carrying resistance only to kanamycin (50 µg/mL) and not
to ampicillin. This led us to suspect
that our construct might indeed be present in these colonies.

To verify this, a colony that exhibited only kanamycin resistance
(50 µg/mL) was selected. DNA plasmid
extraction and purification were performed using the PureLink™
Quick
Plasmid Miniprep Kit (Invitrogen™,
Lot #01260760). Subsequently, enzymatic digestion was carried out
to
linearize the DNA, followed by
agarose gel electrophoresis for result visualization.

### Evaluation of the resulting constructs

Using SnapGene® software, a simulation of enzymatic digestion was
conducted on the constructs to identify
potential enzymes for linearization. Subsequently, in the
laboratory, the digestion reaction of the three
constructs LysK, LysCSA13, and LysSS was performed using SmaI and
EcoRI restriction enzymes. LysK was
linearized by the SmaI enzyme, while both LysCSA13 and LysSS were
linearized by EcoRI (Figure 8). All our constructions were according to the expected bands.5,7,10,11

![](https://static.igem.wiki/teams/4607/wiki/parts/agarosas/todas-las-agarosas-v22.png)

**Figure 8.** pDNA agarose gel
electrophoresis (1%). (a) Lane 1.
Quick-Load® Purple 1 kb Plus DNA
Ladder. Lane 2. LysK digested with single-cut enzyme SmaI
(2997
bp)
pDNA. Lane 3. Negative control LysK
digestion (2997 bp) pDNA. (b) Lane 4. Quick-Load® Purple 1 kb
Plus
DNA Ladder. Lane 5. LysCSA13 digested
with single-cut enzyme EcoRI. Lane 6. Negative control
LysCSA13
digestion (3159 bp) pDNA. (c) Lane 7.
LysSS digested with single-cut enzyme EcoRI (2892 bp). Lane 8.
Negative control LysSS digestion (2892 bp)
pDNA. Lane 9. Quick-Load® Purple 1 kb Plus DNA Ladder.

## Second Round: Protein Expression

The next step of our recombinant protein production process was
grouped into the second round of
experimentation. All the selection, optimization, and
standardization of our protein induction protocols
were performed as follows:

### Protein production

Our constructs contain the biobrick BBa\_J435350, a T7 promoter
with
LacO regulation regions. It was chosen
because of its high transcription levels, and regulation activity.
To induce the protein production of
our *E. coli* BL21 (DE3) transformants, isopropyl β-D-1
thiogalactopyranoside (IPTG) was used as
inducer for the LacO regulation regions, then the reagent makes
the
transformants available to synthetize
our endolysins.

### Protein induction kinetics

To identify the best protein induction parameters, protein
induction
kinetics were performed. Based on previous studies, we identified
that
the optimal temperature and time conditions were about 6 hours at
37° C.
Related to the IPTG concentration, the experiments were performed
at
0.2 mM,
0.5 mM, and 1 mM, for all of our transformants. The inductions
were
performed
with the *E. coli* BL21 (DE3) transformed with the
expression
plasmids
obtained from the Golden Gate Assembly.

 Protein induction
kinetics were
performed at 37° C, for 6 hours at maximum agitation in an orbital
shaker.
Five treatments were included for each endolysin: Negative control
with *E.
coli* BL21 (DE3), negative control with *E. coli*
BL21
(DE3) transformed
with the endolysin without IPTG induction, and three *E. coli*
BL21 (DE3) transformed
with the endolysin induced at three different concentrations of
IPTG, 0.2 mM, 0.5 mM, and 1 mM.
The samples were collected by hour, obtaining 200 μL, by each
treatment.

 To
identify the best induction parameters, a SDS-PAGE was performed.
Considering that the
molecular weight of LysCSA13-ABD was 35.098 kDa, and
LysK-ABD-SH3B30
weights 28.437 kDa.[8,12,13](#referencia8)
The concentrations of the SDS-PAGE phases were 12% and 6%, for
separator and concentrator
phases respectively. The best parameters were IPTG at 0.2 mM and 5
hours for LysCSA13-ABD,
while LysK-ABD-SH3B30 requires 6 hours at the same IPTG
concentration, results can be seen in Figures 6 &
7.We corroborated that our results related to the band weight were according to Cha *et al.,* (2019), Jeong *et al.,* (2023), and Seijsing *et al.,* (2018). For our case, just 6 hours were required for the protein expression.

![](https://static.igem.wiki/teams/4607/wiki/parts/kinetic-lyscsa13.png)

**Figure 9. LysCSA13-ABD 0.2 mM IPTG
protein induction kinetic.**
 *A) ColorBurst protein ladder. B)
E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysCSA13-ABD
non-inducted, fourth hour. D) E. coli BL21
(DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, fourth hour. E)
E.
coli BL21 (DE3)- LysCSA13-ABD
non-inducted, fifth hour. F) E. coli BL21 (DE3)- LysCSA13-ABD
inducted at 0.2 mM IPTG, fifth hour. G) E.
coli BL21 (DE3)- LysCSA13-ABD non-inducted, sixth hour. H) E.
coli
BL21 (DE3)- LysCSA13-ABD inducted at
0.2 mM IPTG, sixth hour. I) E. coli BL21 (DE3), first hour.*

![](https://static.igem.wiki/teams/4607/wiki/parts/kinetic-lysk.png)

**Figure 10. LysK-ABD-SH3B30 0.2 mM
IPTG
protein induction kinetic.**
 *A) ColorBurst protein ladder.
B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysK-ABD-SH3B30
non-inducted, fourth hour. D) E. coli BL21
(DE3)- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, fourth hour.
E)
E.
coli BL21 (DE3)- LysK-ABD-SH3B30
non-inducted, fifth hour. F) E. coli BL21 (DE3)-
LysK-ABD-SH3B30
inducted at 0.2 mM IPTG, fifth hour. G)
E. coli BL21 (DE3)- LysK-ABD-SH3B30 non-inducted, sixth hour.
H)
E. coli BL21 (DE3)- LysK-ABD-SH3B30
inducted at 0.2 mM IPTG, sixth hour. I) E. coli BL21 (DE3),
first
hour.*

To identify our third fusion endolysin we faced some obstacles because of the principle of catalytic activity and its abilities to destabilize gram-negative bacteria, but thanks to the engineering cycle, it was possible to solve them. For the identification of the PCNP-CecA-LysSS protein, massive protein IPTG induction was performed at 0.2 mM, 0.5 mM, and 1.0 mM concentrations. Once the massive inductions were obtained, the B-PER total protein extraction occurred, allowing us to finally run a SDS-PAGE electrophoresis at 15% and 8% polyacrylamide separator and concentrator phases, respectively. Finally, PCNP-CecA-LysSS (24 kDa) was identified (Figure 11).[10,14](#referencia10)

![](https://static.igem.wiki/teams/4607/wiki/parts/lysssreal2-0.jpg)

**Figure 11. PCNP-CecA-LysSS expression. Visualization through a polyacrilamide gel 15% (SDS-PAGE) results.**
 *A) MWM (iBright). B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- PCNP-CecA-LysSS non-inducted. D) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 0.2 mM IPTG. E) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 0.5 mM IPTG. F) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 1.0 mM IPTG*

### Insouble and soluble protein fractions

Once the best protein induction parameters were established, a new
SDS-PAGE was performed to assess the
protein expression site in the cell. Our endolysins were induced
at
0.2 mM IPTG, for 5 (LysK-ABD-SH3B30)
and 6 (LysCSA13-ABD) hours at 37° C. We obtained a massive protein
expression after pellets were
harvested. To evaluate the expression site, the reagent B-PER
extracted the intracellular proteins,
liberating the soluble and insoluble proteins. For the
LysCSA13-ABD
an insoluble fraction has been
demonstrated (Figure 12). On the other hand, LysK-ABD-SH3B30
yielded
typical results of a soluble protein
(Figure 13).

![](https://static.igem.wiki/teams/4607/wiki/parts/csa13insolsol-para-wiki2-0.jpg)

**Figure 12. LysCSA13-ABD (A) soluble
and (B) insoluble
fractions.**
 *A) Both gels presented the
same distribution of wells, each one corresponding to the
fraction (soluble or insoluble) assigned. A)
MWM (for gel A: Precision Plus Protein Dual Color Standards
(10–250 kD), for gel B: PageRuler™ Plus
Prestained Protein Ladder, 10 to 250 kDa). B) E. coli BL21
(DE3). C) E. coli BL21 (DE3)- LysCSA13-ABD
non-induced. D) E. coli BL21 (DE3)- LysCSA13-ABD induced at
0.2
mM IPTG, 2 μL. E) E. coli BL21 (DE3)-
LysCSA13-ABD induced at 0.2 mM IPTG, 4 μL. F) E. coli BL21
(DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 6
μL. G) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM
IPTG,
8 μL. H) E. coli BL21 (DE3)-
LysCSA13-ABD induced at 0.2 mM IPTG, 10 μL. I) E. coli BL21
(DE3)- LysCSA13-ABD induced at 0.2 mM IPTG,
15 μL. J) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM
IPTG, 20 μL.*

![](https://static.igem.wiki/teams/4607/wiki/parts/lysk-ambasfracciones.png)

**Figure 13. LysK-ABD-SH3B30 (A)
soluble
and (B) insoluble
fractions.** *Both gels presented the
same distribution of wells, each one corresponding to the
fraction (soluble or insoluble) assigned. A)
MWM (for gel A: Precision Plus Protein Dual Color Standards
(10–250 kD), for gel B: PageRuler™ Plus
Prestained Protein Ladder, 10 to 250 kDa). B) E. coli BL21
(DE3). C) E. coli BL21 (DE3)- LysK-ABD-SH3B30
non-induced. D) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at
0.2 mM IPTG, 2 μL. E) E. coli BL21 (DE3)-
LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 4 μL. F) E. coli BL21
(DE3)-LysK-ABD-SH3B30 induced at 0.2 mM
IPTG, 6 μL. G) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at
0.2 mM IPTG, 8 μL. H) E. coli BL21 (DE3)-
LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 10 μL. I) E. coli BL21
(DE3)- LysK-ABD-SH3B30 induced at 0.2 mM
IPTG, 15 μL. J) E. coli BL21 (DE3)-LysK-ABD-SH3B30 induced at
0.2 mM IPTG, 20 μL.*

## Third Round: Protein Purification

The current section emphasizes on the obtaining of the specific fusion-endolysins, separating them from the crude protein extracts. This is possible because of the previous *in silico* design, in which we incorporated a 6X HisTag to its purification by using a metal ion affinity chromatography column.

To perform the protein purification, we followed the protocol described in the Experiments Page. For the case of the LysK protein, we correctly identified the purified protein through a 12% polyacrylamide electrophoresis SDS-PAGE by using our soluble fraction collected from the crude protein, which was inducted at 0.2 mM IPTG at the optimal conditions that are described in the previous sections of this page. The protein was identified as is shown in Figure 14.

![](https://static.igem.wiki/teams/4607/wiki/lysk-pur.jpg)

**Figure 14. LysK purification.** *A) PageRuler™ Plus Prestained Protein Ladder. B) E. coli BL21 (DE3). C) E. coli BL21 (DE3) LysK- non-inducted. D) Wash buffer 1. E) Wash buffer 2. F) Wash buffer 3. G) Elution buffer 1. H) Elution buffer 2. I) Elution buffer 3.*

The LysK protein purification was performed successfully, thanks to this was possible to determine our future experimental designs, and math model parameters. We identified a protein weight band bigger than expected (28 kDa), but comparing our results with the previous studies, we found protein identity and purity in the constructions that contain amidase-2 domains, and those that conserve truncated proteins could present prominent additional bands.[6-9](#referencia6) On the other hand, the LysCSA13 protein purification did not reveal any expected band; it is attributed to the solubilization process, in which no protein appeared.

## Fourth Round: Bactericidal Assays

For this stage the main purpose was to corroborate the catalytic activity of our endolysins, the optimal parameters, and the real effects for each bacteria. In the same way, the chitosan-alginate delivery drug system was analyzed simultaneously with our enzymatic treatment.

Considering our protein purification results, we decided to work on the development of our treatment by using our crude protein extracts. To identify our optimal composition, we designed an experimental model that combines simultaneous enzyme analysis and its effects on the pathogenic bacteria. The full protocol is described on our Experiments Page. Our ELISA plate design was prepared as follows:

![](https://static.igem.wiki/teams/4607/wiki/plate1-enzymes.jpg)

**Figure 1. Distribution of ELISA plates for bactericidal assays.**

According to the previous figure, all the raw endolysin extract wells contain not only the enzyme combination, but also 90 uL of the pathogenic bacterium desired. In our design, a range of 3 rows is requiered for each repetition, and at least 3 are required for the experimental validation (for each bacterium). To evaluate our results, we decided to apply the Response Surface Methodology, which is deeply described in our Model Page as well as our bactericidal assay results.

![](https://static.igem.wiki/teams/4607/wiki/placaquitosan.jpg)

**Figure 2. Distribution of ELISA plates for bactericidal assays with loaded protein chitosan alginate beads.**

To evaluate the effect of chitosan-alginate protein loaded beads and the raw extract protein treatment we identified the following results:

![](https://static.igem.wiki/teams/4607/wiki/saureuscht.jpg)

**Figure 3.** Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, *S. aureus.*

According to the previous figure, all the raw endolysin extract wells contains not only the enzyme combination, but also 90 uL from the pathogenic bacterium desired. In our design, a range of 3 rows is requiered for each repetition, and at least 3 are required for the experimental validation (For each bacteria). To evaluate our results, we decided to apply the Response Surface Methodology, which is deeply described in our [Model Page](https://2023.igem.wiki/tec-chihuahua/model).

 For the validation of our protein-loaded chitosan alginate beads the experimental design was prepared as follows:

![](https://static.igem.wiki/teams/4607/wiki/sagalcht.jpg)

**Figure 4.** Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, against *S. agalactie.*

![](https://static.igem.wiki/teams/4607/wiki/ecolicht.jpg)

**Figure 5.** Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, against *E. coli.*

As can be appreciated in Figures 3 to 5, the addition of the chitosan-alginate beads increased the bactericidal activity for each bacterium, a fact that couldn't be completely developed without the stakeholder's suggestions.

[Proof of concept

Visit our **Proof of Concept Page** to learn more about our testing phase!](proof)

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