iGEM 2023 — Team unimuenster

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# Notebook

## 12

### Dec

##### Here you can find the progress reports of our project. Follow us in every step of our work!

## 2022

### - December -

#### Calendar week 50

A first meeting with Theresa Wörmann and Lasse Middendorf of
last year‘s iGEM team took place to exchange ideas and start
to plan the formation of this year‘s team. Stefanie, Emelie
and Dennis K. got together as this year‘s student leaders.

#### Calendar week 51

A meeting with our PI Prof. Jochen Schmid was held to discuss
the next steps on the way to a new team. Afterwards, an
information event took place to recruit potential iGEMers.

#### Calendar week 52

Up until December 31st, future team members applied
via e-mail.

## 2023

### - January -

#### Calendar week 1

**General matters:** Applications were reviewed,
and the student leaders talked to some of the applicants to
determine the most dedicated ones.

#### Calendar week 2

**General matters:** The first team meeting was
planned, and the next steps were discussed with our PI.

#### Calendar week 3

**General matters:** We held our first team
meeting, beginning to get to know each other as there were
only a few members of our diverse team that already worked or
studied together.
[Our team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
consisted of ten MSc Biotechnology students, nine BSc Bio
Sciences students, one MSc Mathematics student, one BSc
Physics student, and one BSc Informatics student, adding up to
22 highly motivated students.

#### Calendar week 4

**General matters:** We established a weekly team
meeting and divided into six subgroups for further work
unrelated to the wetlab: Sponsoring/Finance, Organization,
Social Media,
[Education](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)/[Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D), Teambuilding, and Wiki.

#### Calendar week 5

**General matters:** We started our search for a
project by splitting into duos. These duos researched
potential projects and prepared short presentations.

**Sponsoring:** In the first weekly meeting, a
general division of work was developed, and the sponsorship
folder was designed.

**Organization:** The contact with the REACH
EUREGIO Start-Up Center was established, and we brainstormed
potential working fields.

**Education:** We decided on several future
projects we will want to pursue: a podcast, school visits, and
visits to other institutions.

## - February -

#### Calendar week 6

**General matters:** The first potential project
presentation was held. We cleaned up and decluttered our iGEM
office in the Institute of Molecular Microbiology and
Biotechnology of the University of Münster.

**Fundraising:** The texts for the sponsoring
folder were written, and photos and citations of each team
member were integrated.

**Organization:** A vacation plan was developed
to maintain the overview about absent team members throughout
the year.

**Education:** Research for the first few
episodes of our podcast started.

#### Calendar week 7

**General matters:** Three new project
propositions were presented. In addition, our Principal
Investigator Prof. Dr. Ing. Jochen Schmid and our first
Project Instructor Pia Lanvers introduced themselves to our
whole team.

**Sponsoring:** Last improvements of the
sponsoring folder texts were realized, and the sponsoring
packages available for potential sponsors were developed.

**Organization:** Planning of the research
weekend began.

**Social Media:** A first Instagram post
presenting our team was posted.

**Education:** Research of the first podcast
episode was finished.

#### Calendar week 8

**General matters:** Five additional projects
were presented, and Theresa and Lasse were introduced as
Project Advisors as well as Maximilian Zander as a second
Project Instructor. A meeting with members of the last team
was held to exchange some ideas and thoughts with them.

**Fundraising:** Design of the sponsoring folder
was finished, and texts for the OnePager were redacted.

**Organization:** Plans for the research weekend
were finalized.

#### Research weekend

On February 26th and 27th, we organized a research weekend, during which we informed ourselves about the thirteen project propositions we held over the course of the last weeks. Finally, we decided on three projects we wanted to present to an expert audience.

#### Calendar week 9

##### Student Leaders:

Invitations for experts to attend our final project decision meeting on March 13th.

##### Fundraising:

The sponsoring folder was sent to Jana Rohlfing of the Finance and Controlling department of the University of Münster and Prof. Dr. Johannes Wessels, the Rector of our University for further inspection.

##### Organization:

A list of the medal criteria was put together.

##### Social Media:

Posts regarding the research weekend and a short introduction of the iGEM competition itself were posted.

##### Education:

Hardware for the recording of the first episode of our Podcast was procured.

## - March -

#### Calendar week 10

**Student Leaders:** Coordination of the feedback
regarding our proposition of projects in front of several
professors and other experts.

**Sponsoring:** Work on the OnePager continued.

**Social Media:** Post of the research weekend
was posted, and a large Instagram group with all iGEM teams
that will be at the Grand Jamboree was joined.

**Education:** The first episode of our podcast
"GENial" was recorded, and research for the second one
continued.

#### Calendar week 11

**General matters:** We presented the three
projects we voted for on the research weekend to an audience
of experts and thereafter decided on the one we wanted to
continue:
[BeeVAX](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D), a method of vaccinating honey bees against an
entomopathogenic fungus that can then be brought out to combat
*Varroa* mite infestation without harming the bees.

**Student Leaders:** A meeting with our PI was
held to gain further insights on how to continue to work on
our freshly elected project by taking it into the wet lab.

**Fundraising:** The sponsoring folder was
finalized, and a meeting with Jana Rohlfing took place to
inform ourselves about the tax policy of the university. A
list of potential sponsors was prepared as well as a script
for sponsoring calls.

**Organization:** A list of the existing
materials, chemicals, glassware, etc. in our lab was started.

**Education:** The first episode of our podcast
was cut, and a Spotify account to post it on was created.

#### Calendar week 12

**General matters:** The whole team started to
research aspects of our project, and a provisional lab plan was
created.

**Fundraising:** The first calls of potential
sponsors proved to be partially successful: several lab
equipment companies agreed to sponsor materials, but as of then,
no financial sponsoring could be secured.

**Organization:** The work on the inventory list
continued.

**Social Media:** Several posts regarding the
research weekend, our podcast, sponsoring, and synthetic biology
were posted.

**Education:** The first episode of the podcast was
published on Spotify.

#### Calendar week 13

**General matters:** A more detailed lab plan was
made, and necessities of each of the four newly created lab
groups, Expression of Vitellogenin, Binding assay, Directed
Evolution, and Rational design, were collected in a future list
of orders. The planning for the Junior Jam started with an
approximate timetable and the search for suitable locations.

**Social Media:** The immw (iGEM Münster Meme
Wednesday) was started.

## - April -

#### Calendar week 14

**Fundraising:** Calling potential sponsors
continued as well as searching for new ones.

**Organization:** A first ordering list was
finalized.

**Education:** Planning of the school visits
began, and the second episode of our podcast was recorded and
cut.

**Expression of Vitellogenin:**
*Komagataella phaffii* was decided on as an optimal
expression organism, as it uses several methods that ensure
the protein will be correctly folded and post-translationally
modified.

#### Calendar week 15

**General matters:** A MiniModule was planned by
the Master students to teach the Bachelor students and all
non-biology students some fundamental experiments commonly
used in molecular biology.

**Organization:** Research for electronic lab
books showed eLabnext to be an optimal solution to maintain
the overview about all four of our wet lab groups.

**Education:** A senior citizen home in Münster
was contacted and expressed their interest in having us there
to teach the elderly residents a little about synthetic
biology.

**Expression of Vitellogenin:** Research was
finished, and definite experiment plans for the first weeks
were made.

#### Calendar week 16

**General matters:** We officially registered for
the iGEM competition. The first order of the
*vitellogenin* sequence and some primers were ordered,
and the first materials from New England Biolabs arrived. A
first speaker for the Junior Jam was found: our PI Prof. Dr.
Ing. Jochen Schmid. A lecture room in the castle of Münster
was booked.

**Fundraising:** First material sponsorings were
approved.

**Social Media:** The second episode of our
podcast was uploaded.

**Education:** Visits of schools and retirement
homes were further planned out.

**Expression of Vitellogenin:** Plasmid maps and
Primers were created, and the workflow was discussed with our
Project Instructor Max Zander.

**Rational design:** Research for a docking
software was started.

#### Calendar week 17

**General matters:** On April 24th, one of our
three Student Leaders, Dennis Kloster, decided to end his work
on iGEM due to high levels of stress caused by the
simultaneous work for his Bachelor studies, iGEM, and his
courses of study’s student council.

**Expression of Vitellogenin:**
[Media and buffers](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
were prepared, and new primers for the work with the iGEM
distribution kit were ordered.

**Rational design:** Research continued in the
fields of structure prediction/ binding site prediction.

## - May -

#### Calendar week 18

**General matters:** As a replacement for Dennis
Kloster, we unanimously elected Emil Beurer to serve as our
third Student Leader.

**Fundraising:** Shipments from Roth and Sarstedt
arrived.

**Expression of Vitellogenin:** Work in the wet
lab started by amplifying the genes necessary for the assembly
of our plasmid. The distribution plate proved to be mislabeled
for two of the three genes.

**Rational design:** Research for fitting
bioinformatics tools to gain information about the binding
sites was continued.

#### Meet-up Dusseldorf

On May 6th, we met iGEM Düsseldorf and iGEM Aachen for a brief
[meet-up](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27community%27%29%20%7D%7D)
in Dusseldorf, discussing our project ideas and exchanging
potential collaborations as all of us will work with unicellular
fungi.

#### Calendar week 19

**General matters:** The REACH confirmed to do
two workshops to improve pitch and start-up focus of the iGEM
teams participating on our meet-up.

**Fundraising:** Shipments from Eppendorf and
Brand arrived, and we talked with the foundation "Westfälische
Landwirtschaft" about supporting our project.

**Wiki:** A preliminary distribution of tasks
concerning the design was made.

#### LABvolution

On May 10th, our team visited the LABvolution, the biggest
laboratory equipment fair in Germany, where we got in personal
contact with some of our supporters and supporters-to-be.

#### Calendar week 20

**General matters:** The ASTA Münster (general
student committee) confirmed to grant 700 € for our meet-up.
Indico was set up for coordinating the registrations.

**Fundraising:** Conversations with AGRAVIS as a
potential main sponsor began.

**Education:** The third episode of our podcast
aired, and a meeting with a local beekeeper’s association was
planned.

**Expression of Vitellogenin:** As our previous
plasmids continued to show wrong bands on our gel
electrophoreses, we started to search and ask for new ones we
could use in several institutes of our university.

**Rational design:** Efficient rational design
proved to be ineffective because of the unknown binding sites
and the huge size of the protein. Therefore, the rational
design lab group was fused with the directed evolution group
to form rational mutagenesis.

#### Calendar week 21

**Fundraising:** Several small shipments arrived,
and a meeting with a representative from Eppendorf was held to
discuss the possibility of obtaining a license for the
eLabnext for our team.

**Expression of Vitellogenin:** As the search for
adequate plasmids continued, plans for a potential side
project in the form of an auxotrophic
*K. phaffii* strain were made.

#### Calendar week 22

**General matters:** Another research day was
done to get more into detail with our project ideas, lab work,
and most importantly how we could imagine the implementation
of our research.

**Organization:** The whitelist was checked for
the parts we will want to use in our project, and the
preliminary safety form was started.

### - June -

#### Calendar week 23

**General matters:** This week we voted on what
village we wanted to join with our project. We decided to
register for Conservation as our first choice and
Bioremediation and Agriculture as backup plans. A new subgroup
was invented to tackle the issues of the upcoming months:
*Storytelling*. A second speaker for the Junior Jam was
recruited: Prof. Dr. Andreas Möglich.

**Fundraising:** The sponsoring conversations
with AGRAVIS failed, therefore we were still in dire need of
monetary sponsoring partners.

**Education:** The visit of the beekeeper’s
association took place, from which we gained valuable insights
into the work of a beekeeper in Germany. Additionally, the
idea of a child book first came up and detailed plans for the
visits of the retirement home and youth group were finalized.

**Storytelling:** The first meeting laid a
foundation for future fields of work of this subgroup which
includes the Promotion video, presentations for the meet-up
and Grand Jamboree.

**Binding assay:** Planning of the PAMP
extraction started and an ordering list was prepared.

**Rational mutagenesis:** Some homologous protein
sequences from solitary bees were selected to be tested for
PAMP binding affinity analysis. A vector for the yeast surface
display was ordered.

#### Calendar week 24

**General matters:** PhD Sonja Billerbeck
confirmed to work as a third speaker on our Junior Jam, and
the registration opened for all teams.

**Fundraising:** A meeting with the foundation
„Westfälische Landschaft“ resulted in a sponsoring contract of
5000 €.

**Organization:** The preliminary safety form and
check-in forms for *K. phaffii*, *vitellogenin*,
and *Saccharomyces cerevisiae* were submitted.

**Education:** The first batch of interview
invitations for important researchers and stake holders for
our project were sent out.

#### Calendar week 25

**General matters:** This weekend, we went on a
camping trip to increase our team efforts by getting to know
each other even better and also non-work-related.

**Fundraising:** Another 5000 € were obtained
through funding projects of our university.

**Education:** A question list for expert/stake
holder interviews was created.

**Storytelling:** The preliminary project
description was redacted and posted on the team wiki webpage.

**Expression of Vitellogenin:** Several attempts
to build the vector using different backbones failed. The
research for the CRISPR side project for making an auxotrophic
*K. phaffii* strain.

**Rational mutagenesis:** The rest of the
materials were ordered. Highly conservated areas were detected
by aligning multiple homologous sequences.

#### Calendar week 26

**General matters:** Several meetings with the
bureaucratic representative of our uni, Lars Fischer, were
held to discuss matters such as fire protection and room
booking. In addition, we booked a food truck and informed
ourselves about electricity availability in our locations.

**Education:** The fourth episode of our podcast
aired.

**Wiki:** Picked up work by familiarizing
ourselves with the Gitlab and coding.

**Storytelling:** Work on the promotion video
started, a preliminary screenplay was written.

**Binding assay:** We held three meetings with
experts from our uni that work with protein purification and
biopolymers.

### - July -

#### Calendar week 27

**Fundraising:** We won the iGEM Impact Grant and
received money from the university for organizing student
course modules. (10.000 € total)

#### Calendar week 28

**Organization:** We set up our new lab book,
eLabnext, with our current inventory list, order lists,
protocols, and lab book entries.

**Expression of Vitellogenin:** Two of our
plasmids that tested positive in the colony PCR were sent to
be sequenced.

#### Calendar week 29

**General matters:** This week we stayed at the
marine biological station in Carolinensiel, where we started
working on the wiki texts, revised some of our lab plans, and
did some teambuilding activities. The registration for the
Junior Jam concluded; in total, 103 iGEMers of 19 teams coming
from six different European countries will visit us.

**Wiki:** In Carolinensiel, we started to learn
some basic HTML/CSS coding.

**Expression of Vitellogenin:** Research for a
second side project,
[CTL14](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D),
began in Carolinensiel.

#### Calendar week 30

**Wiki:** A color palette was invented, and ideas
for the team page were collected.

**Rational mutagenesis:** Building of the vector
for the yeast surface display was started.

#### Calendar week 31

**Fundraising:** The financial plan of our
project was revised.

**Rational mutagenesis:** The building of the
vector was completed, and the yeast strain for the yeast
surface display was ordered.

### - August -

#### Junior Jam: The European Meet-Up

From 4th to 6th of August, we invited iGEM Teams from all over
Europe to participate in our “Junior Jam: The European Meet-Up”.
Next to connecting and socializing with other teams, learning
about the projects and approaches of them was the main goal of
our meet-up.

#### Calendar week 32

**Organization:** Attribution form was prepared.

**Social Media:** LinkedIn account was created.

**Education:** Fifth episode of the podcast
aired.

**Wiki:** Work on the wiki started by adjusting
the header, footer, and side bar.

**Inclusivity:** We planned our
[pipette](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27inclusivity%27%29%20%7D%7D)
for people with visual impairments.

**Expression of Vitellogenin:** We switched from
*Escherichia coli* turbo to *E. coli* Top10 due
to *E. coli* turbo's trait to multimerize plasmids.

**Rational mutagenesis:** We built four
expression vectors for the yeast surface display.

#### Calendar week 33

**General matters:** Unfortunately, we lost one
student member, Sven Spehr, due to inactivity. We actively
started to write wiki texts. From this week on, 5-6 texts were
finished every week.

**Storytelling:** Work on the
[Promotion Video](https://m.youtube.com/watch?si=6AzMb7axn44fOhTo&v=Pgiq9xqqUPg&feature=youtu.be)
started.

#### Calendar week 34

**Education:** Seventh episode of our podcast
aired.

**Storytelling:** Last shots for the Promotion
video were filmed and animations were designed.

**Rational mutagenesis:** We ordered a new yeast
strain because the previously ordered ones wouldn't grow.
Docking simulations were conducted.

#### Calendar week 35

**General matters:** We chose responsible persons
for every wiki page to accelerate wiki development.

**Education:** Interview with Dr. Maus took
place.

**Wiki:** Footer was designed.

**Expression of Vitellogenin:** We ordered
fragments to assemble into an expression vector via Gibson
Assembly.

**Rational mutagenesis:** Modeling of chimaera
proteins to determine stability and feasibility.

### - September -

#### Calendar week 36

**Education:** The youth group and retirement
home visit took place, and another interview was done.

**Storytelling:** Promotion video is done
producing.

**Expression of Vitellogenin:** The assembly was
successful, we have our
[ready-to-use plasmid](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D).

#### Calendar week 37

**General matters:** We had our first pitch
training session offered by the REACH and changed our approach
on the presentation accordingly.

**Organization:** Final safety form was filled
in.

**Storytelling:** Description and subtitles for
Promotion video are done.

**Inclusivity:** The interviews with the school
for the blind and the association for the blind were held.

**Rational mutagenesis:** First and second FACS
run was done and showed
[results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

#### Calendar week 38

**General matters:** A first draft for our
village presentation was prepared.

**Organization:** We planned a wiki weekend for
correction, writing, and coding our website.

**Education:** We visited the high school and
museum, and our eighth podcast episode aired.

**Storytelling:** A first concept for the landing
page was elaborated.

**Inclusivity:** A lab run with simulated visual
impairments was done.

**Rational mutagenesis:** Preparation of the
library and blue-white selection to determine the mutation
rate were done.

#### Calendar week 39

**General matters:** The wiki weekend proved to
be rather effective; the majority of our texts are ready for
external corrections.

**Rational mutagenesis:** We did our last FACS
run which yielded no usable results.

### - October -

#### Calendar week 40

**General matters:** We invested all our energy
in writing and correcting wiki texts and building our website.

[Community](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27community%27%29%20%7D%7D)

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

{% include 'footer.html' %}

{% extends "layout.html" %} {% block header %}

# {{ self.title() }}

{% endblock %} {% block title %}Engineering Success{% endblock %} {% block
sidebar %}
[##### {{ self.title() }}](#summary)

[Introduction](#section-1)
[Best Binding Affinity to Zymosan](#section-2)
[Predicting Zymosan Binding in Vitellogenin Models](#section-3)
[Enabling the Expression of Vitellogenin](#section-4)
[References](#section-5)

{% endblock %} {% block page\_content %}

## Overview

Designing a project as complex as BeeVAX involves extensive planning and
continually refining those plans to achieve optimal outcomes.

Our project aimed to create an enhanced version of Vitellogenin via protein
engineering. To achieve this goal, we designed our experiments within each
lab group in both, dry lab and wet lab. We were very excited to apply the
plan to real laboratory conditions and test if our design worked out as
intended. Throughout this process, failures, whether minor or major, were a
challenge for our motivation. Nevertheless, these challenges often became
opportunities to improve our design and learn something for future attempts.
We believe that this is the essence of engineering success. In the
following, we present three examples that passed every step of the
engineering cycle to contribute to an optimized version of Vitellogenin: 1)
Investigating the best binding affinity to zymosan, 2) Predicting zymosan
binding in Vitellogenin models and 3) Enabling the expression of
Vitellogenin.

> "The human foot is a masterpiece of engineering and a work of art."
>
> - Leonardo Da Vinci

## Introduction

"The human foot is a masterpiece of engineering and a work of art." More
broadly you could say nature in general is an evolutionary masterpiece of
engineering and we are the ones to learn from it. As bioengineers we apply our
knowledge about biological mechanisms and systems to create new innovative
systems. Thereby, we can enable solutions for unsolved problems or invent new
products using the inherent power of nature itself.

Our project is a middle-out approach combining forward and reverse
bioengineering to generate the bee's egg-yolk protein Vitellogenin with an
optimized binding affinity against fungi PAMPs to enable a new treatment
against the *Varroa* mite infestation of bee hives. The reverse
engineering approach is based on the aim to gain more knowledge about the
binding sites and domains of *Apis mellifera*`s Vitellogenin. In the
forward engineering steps, we focused on increasing the binding affinity of
Vitellogenin to fungi's PAMPS by a random mutagenesis-based approach, more
precisely, the directed evolution of the protein combined with a yeast surface
display (YSD).

To successfully engineer our protein of interest, we passed through the
engineering cycle in different parts of our project. The cycle consists of
four successive steps: Design- Build- Test- Learn. During the Design and Build
step, the known and unknown parts of the projects are estimated and
implemented in a detailed lab protocol. By testing, the design is approved or
qualified for optimization. The latter leads to the following step: Learn,
which enables a re-design and optimization of the project design.

![The graphic shows the model of an engineering cycle containing four steps: Design, Build, Test and Learn. The first step, Design, contains the following information: Planning which materials, methods, and steps to apply to reach the aims of your initial ideation. The second step, Build, contains the following information: Implement the design by creating the connected biological system. The third step, Test, contains the following information: Measure and check your created system if it works the way you designed it. The last step contains the following information: Analyse your test results and take advantage of it to eventually re- design your system and improve it. After the four steps an arrow indicates that the cycle starts again in the next design cycle.](https://static.igem.wiki/teams/4675/wiki/engineering/fig1.png)

Figure 1: Model of the engineering cycle with the four steps Design, Build,
Test and Learn.

Each part of the project passed through several cycles to finally reach the
obtained aims. But looking back, also the whole project itself went through
various of these cycles. In the following, some aspects of the engineering
process of our project are depicted. It is important to consider that an
engineering process is only possible with a previous failure which is erased
by finding the weak point and optimizing the project design by starting a new
cycle.

---

## Investigating the Best Binding Affinity to Zymosan for YSD

One principal part of our project was the use of a yeast surface display (YSD)
to present the domains of the *Apis mellifera*`s Vitellogenin on the
surface of  *Saccharomyces cerevisiae*. To read more about the
constructs visit the
[project description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D). The cells of the YSD were analyzed using fluorescence activated cell
sorting (FACS) with the two following main aims: 1) to investigate which
domain of Vitellogenin shows the highest zymosan binding affinity and 2) the
cell sorting of error-prone PCR mutated versions of these domains to find
mutations which lead to an increased binding affinity to zymosan.

This is the first attempt measuring zymosan binding to Vitellogenin domains
via a YSD. Therefore, no protocols regarding the handling of zymosan and
testing of the binding affinity using a YSD based approach were available. We
were required to depend on YSD protocols employing diverse binding substrates,
commonly utilized in antibody engineering, to assess an appropriate
methodology. For antibody engineering the substrates are mainly biotinylated
[1][2]. Hence, these approaches do not include fluorescence marked substrates
and are inherently different from ours.

![The graphic shows two engineering cycles for the best binding affinity for zymosan in the YSD. It starts at Design 1 with the following information: Adopting FACS washing protocols to bee Vitellogenin and zymosan. The next step is Build 1 with the following information: 500 microliter of PBS and 35° Celsius are incubated for 5 minutes and centrifugated at 13,000xg. The next step is Test 1 with the following information: After the washing steps the antibody signal of the constructs is decreased. The next step is Learn 1 with the following information: To adjust the zymosan concentration washing steps are not sufficient. Then, the second cycle begins. The step Design 2 contains the following information: Plan to test the binding kinetics of bee Vitellogenin to zymosan. The next step is Build 2 with the following information: Determine which concentrations are needed for a kinetic calculation. The next step is Test 2 with the following information: Realizing the zymosan rate is reached earlier than expected. The last step is Learn 2 with the following information: The dissociation constant of 0.61 microgram per microliter determines the right zymosan concentration.](https://static.igem.wiki/teams/4675/wiki/engineering/fig2.png)

Figure 2: Schematic illustration of the two engineering cycles to find the
best binding affinity to zymosan for YSD.

### Cycle I

**Design & Build I:** To find the best binding affinity, the
zymosan had to be fragmented and labeled with a fluorescence marked molecule,
Fluorescein-5-thiosemicarbazide (5-FTSC). 5-FTSC has an absorbance of
492 nm, and an emission of 516 nm. 1 mg/mL of the labeled
fragments were solved in PBS buffer and the cells were incubated in 100
of  ared mixture for 60 minutes. The final volume contained 500 µL
of phosphate buffered saline (PBS) with 2x106
*Saccharomyces cerevisiae* EBY100 cells displaying the four
different constructs. The use of 1 mg of labeled zymosan was an estimated
amount. Thereby, we wanted to ensure that the Vitellogenin domains on the
surface of the cells are oversaturated. Afterwards, the cells were washed
according to the following protocol: Each washing step included the
centrifugation of the sample at 13,000 x g for 1 minute and resuspension of
the cell pellet in 500 µL cold PBS buffer. PBS buffer is widely used to
remove unbound and excessive compounds in enzyme immunoassays [3]. Following,
the samples were incubated at 35 °C for 5 minutes. The temperature of 35°
was based on the natural hive temperature of Western honey bees [4]. After
every step the sample was analyzed in the FACS because we wanted to estimate
the required amount of washing steps.

**Test I:** During the FACS run we performed three washing steps
for every sample. The total amount was limited to a third wash because
afterwards the cell counts in the FACS decreased drastically. So, there were
not enough cells for a fourth run.

The FACS results were analyzed with the single cell flow cytometry FlowJo.
The first washing step showed a lower red fluorescence at a wavelength of 633
 nm than the unwashed samples. Fig. 3 shows the decrease for three
different zymosan concentrations of the construct displaying the
α‑helical+DUF1943 domain. During the analysis we focused on the
α‑helical+DUF1943 because it showed the best binding affinity to zymosan. The
red fluorescence corresponds to the cMyc antibody binding of the construct.
Therefore, it is independent from the zymosan binding and should not change
throughout the washing steps. But apparently, the washing steps were too harsh
for the low binding affinity of the cMyc antibody to the cMyc-tag.
Additionally, the washing steps did not wash off the zymosan as indicated in
Fig.4.

![The figure shows a graph chart with three graphs representing three different tested zymosan concentrations. The x-axis only shows to points: pre wash and post wash. The y-axis shows the percentage of cells in the `high display` gate. All graphs show a strong decrease after the wash.](https://static.igem.wiki/teams/4675/wiki/engineering/fig3.png)

Figure 3: The red fluorescence signal at 633 nm wavelength before
and after the first washing step, shown for the construct
Vg\_α‑helical+DUF1943 as an illustrative example. A decrease in the
antibody signal was found in all samples with the three different
zymosan concentrations of 0.02 µg/mL, 20 µg/mL and
200 µg/mL.

![The figure shows a graph chart with three graphs representing three different tested zymosan concentrations. The x-axis only shows to points: pre wash and post wash. The y-axis shows the percentage of cells in the `high display` gate. All graphs show are stable before and after the wash.](https://static.igem.wiki/teams/4675/wiki/engineering/fig4.png)

Figure 4: The green fluorescence signal at 517 nm wavelength before
and after the first washing step, shown for the construct
Vg\_α‑helical+DUF1943 as an illustrative example. No significant change
of the zymosan signal was found in all samples with the three different
zymosan concentrations of 0.02 µg/mL, 20 µg/mL and
200 µg/mL.

**Learn I:** With the unexpected results of the first FACS run we
concluded that the washing steps are too harsh for the low binding force of
the cMyc- antibody- antigen- complex. Furthermore, it did not affect the
unbound zymosan concentration. Therefore, we needed to find a new way to
estimate the right amount of zymosan for the measurement.

### Cycle II

**Design & Build II:** Another approach to find the right amount
of zymosan is the calculation of the binding kinetics of zymosan to the
domains of Vitellogenin. Therefore, a normalization of the results with a cell
control is required and a kinetics model fitting is needed. The binding
kinetics lead to a dissociation constant KD which is used to
describe the binding affinity of one molecule to another. Using this approach,
the ideal amount of zymosan for every domain can be estimated.

The concentrations of zymosan have been tested in a range of 0.2 µg/mL –
200  µg/mL. We chose a broad range because we could not disclose it
further. The kinetics become more accurate the more zymosan concentrations
within the range are measured.

**Test II:** During the second FACS run we tested four different
concentrations in increments of 10, 0.2 µg/mL, 2 µg/mL, 20 µg/mL and
200 µg/mL. For the third FACS run the same concentrations were tested and
enlarged by 0.5 µg/mL, 5 µg/mL and 50 µg/mL of zymosan. Unfortunately,
the third FACS run did not lead to any valuable results because the constructs
held deficient inserts. Therefore, we based the calculation of the
dissociation constant KD on a limited number of measuring points.
During the process we decided to focus on the construct Vg\_α-helical+DUF1943
because we observed the best binding affinity for this variant. To read more
about the binding affinity of the constructs and the defects visit the
[results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
page.

**Learn II:** Overall, we were able to determine the KD
of the labeled zymosan and the construct Vg\_α‑helical+DUF1943 which is
0.61 µg/mL. Based on the results we decided to use 2 µg/mL for the
library sorting as they represent low and high saturation points of the
Vg\_α‑helical+DUF1943 construct. Thereby, we bypassed the washing steps and
found a justified concentration of labeled zymosan to use for the YSD
constructs. To obtain a KD
which is even more accurate, a higher number of measuring points should be
considered.

### Outlook

After all, we evaluated a protocol to test the zymosan binding affinity of
Vitellogenin domains in a YSD. Still, more measuring points are required to
further optimize the kinetics and consequently our working protocol. Also, the
incubation time of zymosan with the domain- displaying cells could be adapted
to additionally increase the binding affinity.

---

## Predicting Zymosan Binding in Vitellogenin Models

For our protein engineering approach of Vitellogenin, a binding site
prediction was required. These would allow us to check mutations for improved
binding affinity *in silico* by performing docking simulations.
Unfortunately, there are no papers available that predict concrete binding
sites. So, we decided to perform a modeling of the binding sites for PAMPs as
a dry lab approach.

![The graphic shows two engineering cycles for the prediction of zymosan binding in Vitellogenin. It starts at Design 1 with the following information: Finding a pocket which predicts binding pockets for zymosan. The next step is Build 1 with the following information: Utilization of P2Rank with the AlphaFold structure of Vitellogenin. The next step is Test 1 with the following information: P2Rank indicates 42 binding pocket candidates. The next step is Learn 1 with the following information: P2Rank is not specific enough to focus on certain binding pockets. Then, the second cycle begins. The step Design 2 contains the following information: Finding a method to narrow down the predicted binding pockets. The next step is Build 2 with the following information: AlphaFold mean confidence score and previous research on the domains. The next step is Test 2 with the following information: 4 promising pocket candidates are left. The last step is Learn 2 with the following information: The combination of P2Rank and AlphaFold enclosed the potential binding pockets.](https://static.igem.wiki/teams/4675/wiki/engineering/fig5.png)

Figure 5: Schematic illustration of the two engineering cycles of the
prediction of zymosan binding in Vitellogenin.

### Cycle I

**Design & Build I:** As a first step, we tried to find
computational tools to identify the relevant binding pocket of Vitellogenin.
Most potential tools were not directly accessible and therefore out of
question. In comparison, P2Rank was a tool that was developed with ease of use
in mind and showed the most promising results. It is a stand-alone
template-free tool for prediction of ligand binding sites based on machine
learning [5]. Because no experimental structure of Vitellogenin is available
we utilized the structure predicted by AlphaFold.

In P2Rank, the results are presented in .csv files with a given probability
for every binding site candidate. To visualize the candidates inside the
protein structure, we wrote a
[script](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
for ChimeraX presenting each candidate in different colors and with
corresponding numbers.

**Test I:** The tool P2Rank provided us with 42 predicted binding
pocket candidates for the honey bee's Vitellogenin, with 14 of them having a
predicted probability higher than 10%. For further analysis, we decided to set
10% probability as a threshold.

![The picture shows the protein structure of Vitellogenin which is a huge coil of alpha helices and beta- sheets. The vWF domain can be estimated prominent outside of the protein. The 42 pocket candidates are spread in the main body of the protein without any structure.](https://static.igem.wiki/teams/4675/wiki/engineering/fig6.png)

Figure 6: Vitellogenin structure in ChimeraX, assembled with AlphaFold,
indicating 42 predicted pocket candidates highlighted in different colors.

**Learn I:** Due to Vitellogenin's large size of 1770 amino
acids, we already expected to find many potential pocket candidates.
Additionally, Vitellogenin is known to show many different biological
functions with binding activity to different types of substrates besides PAMP
binding. Thereby, multiple binding sides even indicate the integrity of the
applied tool [6]. Nevertheless, 14 pocket candidates are too many to
appropriately work with.

### Cycle II

**Design & Build II:** Our idea was to narrow down the pocket
candidates by combining other tools with P2Rank to get more information about
the candidates.

Whenever AlphaFold models are used for protein function prediction, the
confidence score (pLDDT - predicted local distance difference test) provided
by AlphaFold should be considered [7]. The pLDDT score is a per-residue
confidence score. On average, the pocket candidates consist of 19 residues.
Therefore, we implemented a
[tool](https://gitlab.igem.org/2023/software-tools/unimuenster)
to add the average confidence score of all involved residues to every pocket
into the .csv file. Moreover, previous research [8] suggested only the
α‑helical and the DUF1943 domains to bind PAMPs. Our own results of the yeast
surface display align with this suggestion. Consequently, we planned to only
consider pocket candidates placed in both domains.

For more insights, visit the
[modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
page.

**Test II:** After calculating the mean confidence score for all
14 pocket candidates of the first cycle, eight pockets showed a confidence
score over 0.85. Four of them were present inside the relevant domains.
Overall, we were able to limit the number of promising pocket candidates to
four, as you can see in Fig. 7.

Table 1: The table shows the probability given by P2Rank for the 14 selected
pocket candidates. The mean\_plddt is the average AlphaFold confidence score
for the involved residues. It represents the combination of P2Rank and
AlphaFold which is required for further narrowing down the pockets

| Name | Rank | Probability | Mean pLDDT |
| --- | --- | --- | --- |
| Pocket 1 | 1 | 0.98 | 90.93 |
| Pocket 2 | 2 | 0.94 | 84.99 |
| Pocket 3 | 3 | 0.92 | 95.61 |
| Pocket 4 | 4 | 0.76 | 92.38 |
| Pocket 5 | 5 | 0.65 | 76.17 |
| Pocket 6 | 6 | 0.47 | 73.23 |
| Pocket 7 | 7 | 0.27 | 84.66 |
| Pocket 8 | 8 | 0.21 | 85.54 |
| Pocket 9 | 9 | 0.18 | 83.89 |
| Pocket 10 | 10 | 0.17 | 89.52 |
| Pocket 11 | 11 | 0.16 | 81.08 |
| Pocket 12 | 12 | 0.13 | 94.65 |
| Pocket 13 | 13 | 0.12 | 95.63 |
| Pocket 14 | 14 | 0.11 | 95.59 |

**Learn II:** If your protein of interest is large and has
multiple proposed functions, additional information is required to make
reasonable predictions of binding sites. The addition of the AlphaFold scores
seems to be a useful complement to that.

### Outlook

Additional research on a potential function of the four binding pockets is
needed to clearly focus on one of them. According to literature [8], candidate
10 (shown in red in Fig.7) seems to be the most promising one. To combine the
findings with the wet lab, we would like to obtain the sequencing of the
improved variants from the directed evolution approach. In the sequencing
data, the location of the mutations can be compared to the location of the
pocket candidates.

![The picture shows the protein structure of Vitellogenin which is a huge coil of alpha helices and beta- sheets. The vWF domain can be estimated prominent outside of the protein. The middle of the protein containing the alpha-helical domain and the DUF domain is highlighted and the 4 promising pockets are present inside the highlighted part.](https://static.igem.wiki/teams/4675/wiki/engineering/fig7.png)

Figure 7: Vitellogenin structure in ChimeraX, assembled with AlphaFold,
indicating four predicted pocket candidates, highlighted in different colors.
The α‑helical domain is presented in turquoise and the DUF1943 domain is
presented in dark blue. Pocket 1 is yellow, pocket 3 is orange, pocket 4 is
hot pink, and lastly, pocket 10 is red.

---

## Enabling the Expression of Vitellogenin

The third engineering example is the encounter and fixing of the plasmid
multimerization in *Escherichia coli* Turbo during the expression
of Vitellogenin. The main contributor to plasmid dimerization and
multimerization is the Recombinase A (RecA), an enzyme active in
recombination. Previous studies were able to observe that RecA is a main
contributor to plasmid dimerization and multimerization, but also
RecA-independent dimerization and multimerization could be observed [9, 10].
During the process of the engineering cycle, we were able to observe decreased
plasmid multimerization in the RecA-deficient *E. coli* Top10,
when compared to *E. coli* Turbo, which has an active RecA ​[11, 12]​.

![The graphic shows two engineering cycles for the Expression system of Vitellogenin. It starts at Design 1 with the following information: In silico design of pSB3CYP_Zeo shuttle vector. The next step is Build 1 with the following information: Construction of the vector in Escherichia coli Turbo. The next step is Test 1 with the following information: The plasmid shows an unexpected large size. The next step is Learn 1 with the following information: Escherichia Turbo multimerizes the plasmids due to RecA activity. Then, the second cycle begins. The step Design 2 contains the following information: Investigation into a recombinase A deficient strain. The next step is Build 2 with the following information: Reconstruction of the plasmids in Escherichia coli Top 10. The next step is Test 2 with the following information: Checking the plasmids for the correct size. The last step is Learn 2 with the following information: Escherichia coli Top10 is deficient of RecA which leads to significant less multimerization.](https://static.igem.wiki/teams/4675/wiki/engineering/fig8.png)

Figure 8: Schematic illustration of the two engineering cycles undertaken for
the construction of the pSB3CYP plasmids and the resolution of the problem of
plasmid dimerization/multimerization.

### Cycle I

**Design & Build I:**  After deciding on
*E. coli* Turbo as the strain in which we wanted to assemble the
construct, we started to design the plasmid for the expression of
*Apis mellifera* Vitellogenin in  *Komagataella phaffii*.
During the assembly of the plasmid, we would construct the shuttle vector
pSB3CYP\_Zeo ([BBa\_K4675009](http://parts.igem.org/Part%3ABBa_K4675009)). The shuttle vector can be used by other iGEM teams for the assembly of
their own expression vectors. Our first aim was to interchange the selection
marker and origin of replication of the pSB3CY\_His ([BBa\_K4188002](http://parts.igem.org/Part%3ABBa_K4188002)) plasmid with ones suitable for *Komagataella phaffii*. After the
assembly of the *zeocinR* gene and *PARS* origin of replication
into the vector, we tried to start with the assembly of the expression
cassette. The *zeocinR* gene and *PARS* were incorporated into
the vector by restriction digestions using the enzymes NotI and SpeI for the
*zeocinR* and SpeI and BamHI for the *PARS*. Next, we assembled
the expression cassette containing the promoter AOX1, terminator AOX1, and
*vitellogenin* via Golden Gate Assembly (RCF [1000]) using the type IIs
restriction enzyme SapI.

**Test I:** Throughout this phase, we tested the vectors and
their intermediates for correct assembly. PCRs and gel electrophoresis were
done to verify the integration of parts into the vector. Interestingly, the
gel electrophoreses of some vectors revealed that they were larger than
anticipated. For example, the constructs of the Lvl2\_plasmids, based on the
pSB3CY\_aeBlue ([BBa\_K4188001](http://parts.igem.org/Part%3ABBa_K4188001)), which were expected to be approximately 6-7 kb in size, were also
observed to be 20 kb in length during gel electrophoresis.

**Learn I:** During this phase of the engineering cycle, we began
to investigate the phenomenon of plasmids appearing larger in size than the
sequence of the plasmids suggests. One of our advisors told us during a
discussion that *E. coli* Turbo has the negative trait of often
dimerizing or even multimerizing plasmids. Indeed, studies showcased that
plasmids often dimerize or multimerize in *E. coli* strains that
are not deficient in Recombinase A (RecA), like *E. coli* Turbo
[9, 10, 12].

### Cycle II

![all relevant infos in caption](https://static.igem.wiki/teams/4675/wiki/engineering/fig9-neuneu.png)

Figure 9: Gel electrophoresis checking for plasmid dimerization in
*E. coli* Turbo and Top10. “Turbo” refers to a plasmid
isolated from *E. coli* Turbo, “Top10” refers to a plasmid
isolated from *E. coli* Top10. The plasmids were tested
based on the pSB3CY\_aeBlue (BBa\_K4188001) with either a tryptophane
synthase (Trp) or uracil synthase (Ura). The plasmid containing the
tryptophane synthase has a length of 6818 bpsp;bp, the one
containing the uracil synthase has a length of 6252 bp. In all
tested plasmids, a second band at 20 kb was observed. These bands
could be the result of dimerized or multimerized plasmids. The bands
corresponding to the multimerized plasmids appear fainter in the Top10
samples.

**Design & Build II:** Based on the knowledge gained in the
Learn I phase, we started to investigate RecA-deficient strains used for
cloning. *E. coli* Top10 is such a strain with a deleted
*recA* gene and should show reduced or no dimerization or
multimerization of plasmids ​[9, 11]​. The construction of the previously
designed plasmid was then resumed as explained above, but with using
*E. coli* Top10 instead of *E. coli* Turbo.

**Test II:** We tested the plasmids again via PCR and gel
electrophoresis to detect possible dimerization and multimerization. Gel
electrophoresis showed that the plasmids based on the pSB3CY\_aeBlue ([BBa\_K4188001](http://parts.igem.org/Part%3ABBa_K4188001)) cloned into *E. coli* Top10 appeared not only at
6-7 kb but also at approximately 20 kb. Comparing the plasmids
from the two *E. coli* strains, we were able to see lighter
bands at 20 kb, possibly indicating less plasmid multimerization
occurring in *E. coli* Top10 (Fig. 2).

**Learn II:** *E. coli* Top10 is a RecA-deficient
strain, and the gel electrophoresis showcased decreased plasmid
dimerization and multimerization occurring during incubation (Fig. 2)
[11]. Studies on recombination in *E. coli* suggest that
recombinases such as RecA support the strand pairing and transfer of
homologous sequences during recombination [13]. Still, we were able to see
plasmid multimerization in plasmids isolated from
*E. coli* Top10, which leads to the assumption that other
factors play a role in plasmid multimerization [10]. Bi et al. were able
to showcase in 1994 that plasmid dimerization can still occur in
RecA-deficient strains. They concluded that RecA-independent recombination
can still occur in homologous sequences of up to 300 bp in length
[14]. Based on this data, we conclude that using
*E. coli* Top10 is beneficial for plasmid cloning when
compared to *E. coli* Turbo, but further optimization has to
be done to decrease the occurrence of plasmid dimerization and
multimerization.

---

## {% include 'animations/arrow.html' %} References

1. [1] Bhupal Ban, Robert C. Blake, Diane A. Blake, “Yeast Surface
   Display Platform for Rapid Selection of an Antibody Library via
   Sequential Counter Antigen Flow Cytometry” (2022) Antibodies, doi:
   10.3390/antib11040061
2. [2] David W. Colby, Brenda A. Kellogg, Christilyn P. Graff, Yik A.
   Yeung, Jeffery S. Swers, K. Dane Wittrup, “Engineering Antibody
   Affinity by Yeast Surface Display” Methods in Enzymology, (2004), doi:
   10.1016/S0076-6879(04)88027-3
3. [3] Mandy Alhajj, Muhammad Zubair, Aisha Farhana, “Enzyme Linked
   Immunosorbent Assay”, National Library of Medicine (2023)
4. [4] Roberto Bava, Fabio Castagna, Christian Piras, Vincenzo Musolino,
   Carmine Lupia, Ernesto Palma, Domenico Britti, Vincenzo Musella,
   “Entomopathogenic Fungi for Pests and Predators Control in Beekeeping”
   (2022) doi: 10.3390/vetsci9020095
5. [5] Radoslav Krivák, David Hoksza,“P2Rank: machine learning based tool
   for rapid and accurate prediction of ligand binding sites from protein
   structure,” Journal of Cheminformatics, Aug. 2018
6. [6] V. Leipart et al., “Structure prediction of honey bee
   vitellogenin: a multi-domain protein important for insect immunity,”
   FEBS Open Bio, vol. 12, no. 1, pp. 51–70, Oct. 2021, doi:
   10.1002/2211-5463.13316.
7. [7] M. Akdel et al., “A structural biology community assessment of
   AlphaFold2 applications,” Nature Structural & Molecular Biology, vol.
   29, no. 11, pp. 1056–1067, Nov. 2022, doi: 10.1038/s41594-022-00849-w.
8. [8] V. Leipart et al., “Identification of 121 variants of honey bee
   Vitellogenin protein sequences with structural differences at
   functional sites,” Protein Science, vol. 31, no. 7, Jun. 2022, doi:
   10.1002/pro.4369.
9. [9] J. R. Bedbrook and F. M. Ausubel, “Recombination between bacterial
   plasmids leading to the formation of plasmid multimers,”
   *Cell*, vol. 9, 4 PT 2, pp. 707-716, 1976, doi:
   10.1016/0092-8674(76)90134-3.
10. [10] R. A. Fishel, A. A. James, and R. Kolodner, “recA-independent
    general genetic recombination of plasmids,” *Nature*, vol. 294,
    no. 5837, pp. 184-186, 1981, doi: 10.1038/294184a0.
11. [11] Genotypes and Genetic Markers of E. coli Competent Cells |
    Thermo Fisher Scientific - DE. [Online]. Available:
    https://www.thermofisher.com/de/de/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/competent-cell-genotypes-genetic-markers.html
    (accessed: Oct. 5, 2023).
12. [12] N. E. Biolabs, NEB® Turbo Competent E. coli (High
    Efficiency) | NEB. [Online]. Available:
    https://www.neb.com/en/products/c2984-neb-turbo-competent-e-coli-high-efficiency#Product%20Information
    (accessed: Oct. 5, 2023).
13. [13] S. C. Kowalczykowski, “Initiation of genetic recombination and
    recombination-dependent replication,”
    *Trends in Biochemical Sciences*, vol. 25, no. 4, pp. 156–165,
    2000, doi: 10.1016/s0968-0004(00)01569-3.
14. [14] X. Bi and L. F. Liu, “recA-independent and recA-dependent
    intramolecular plasmid recombination. Differential homology
    requirement and distance effect,”
    *Journal of Molecular Biology*, vol. 235, no. 2, pp. 414–423,
    1994, doi: 10.1006/jmbi.1994.1002.

[Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

[Measurement](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %} {% block header %}

# {{ self.title() }}

{% endblock %} {% block title %}Experiments{% endblock %} {% block sidebar %}
[##### {{ self.title() }}](#summary)

[Protocols of the experiments](#section-1)

{% endblock %} {% block page\_content %}

## Overview

To accomplish our mission in saving the bees and therefore improve the
immune protein Vitellogenin, we used a lot of different methods and even
designed new protocols to fit our need. More detailed descriptions on how we
worked in the lab are described in the
[results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D). In the PDF below you can find a list of the methods we used to carry out
our experiments.

## Protocols of the experiments:

[Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %} {% block header %}

# {{ self.title() }}

{% endblock %} {% block title %}Results{% endblock %} {% block sidebar%}[##### {{ self.title() }}](#summary)

[Directed Evolution](#section-1)
[Expression of *Helicoverpa armigera* C-type Lectin 14](#section-3)
[Expression of *Apis mellifera* Vitellogenin](#section-5)
[References](#references)

{% endblock %} {% block page\_content %}

## Overview

Western honey bees (*Apis mellifera*) suffer under the influence
of the
*Varroa* mite, traditional control methods bear
[large disadvantages](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
for beekeepers, as they accumulate in the honey and beeswax and spoil the
final product. We wanted to create a new method on how to combat the
*Varroa* mite using the egg yolk protein Vitellogenin.

Since Vitellogenin's involvement in transgenerational immune priming was
only recently discovered [1], there is little information on where and how
the PAMPs are bound to and transported by Vitellogenin. Hence, the heist to
create a Vitellogenin variant optimized for our specific needs was a
challenging task. Where in the protein should we start searching if we don't
know what to look for? Only after heated discussions and extensive research
did we finally develop a comprehensive plan aimed at optimizing Vitellogenin
to facilitate binding to pathogen-associated molecular patterns (PAMPs) from
fungi, thereby enhancing immunity to entomopathogenic fungi, allowing them
to be used to combat *Varroa* mite infestation.

Starting from the wildtype Vitellogenin, we searched for optimized
Vitellogenin in two ways: For the wet lab, we used directed evolution to
introduce mutations into the protein and sort the proteins by their binding
affinity through fluorescence activated cell sorting (FACS). As a dry lab
approach, we did multiple predictions and simulations with individual
Vitellogenin variants from the Western honey bee and other wild bee species.
If we found a variant of Vitellogenin that exhibits the desired properties,
the next step would be to express it in *Komagataella phaffii* and then
test the binding affinity towards fungal PAMPs in a specifically designed
binding assay.

## Directed Evolution

Designing a protein whose mechanism of function is poorly understood presents
numerous challenges, particularly when dealing with a complex protein like
Vitellogenin. Its many different domains and functional regions [1] make it
almost impossible to identify the residues responsible for PAMP (pathogen
associated molecular pattern) affinity, without complex and lengthy trials.
This made the prospect of success using exclusively rational design approaches
limited.

That is why we decided to also pursue a different approach in the form of
directed evolution. For this, a library of sequence variants is generated via
random mutagenesis and is screened with a high throughput screening method to
isolate mutants with the desired properties. The major advantage of this
approach is that no prior knowledge of the proteins mechanisms is needed [2].

Directed evolution has been employed for a wide range of applications
including optimization of wholecell biotechnological processes like
fermentation [3–6], plant breeding [7], protein optimization [2] and
biosensors [8], as well as new opportunities like the prediction of antibiotic
resistances [9, 10] and many more [11, 12]. Its relevance is further
underlined by the award of the Nobel prize in chemistry to Frances H. Arnold
in 2018. She was honoured for developing the first procedures for enzyme
directed evolution.

Many commercial proteins have been optimized via directed evolution. Among
some of the modified parameters are protein stability and activity at
different pH and temperature ranges [13–15], substrate turnover optimization
[16, 17] and substrate specificity [18], but basically all properties of a
protein can be influenced through a fitting directed evolution approach.

As previously discussed, the parameter we needed to improve was the
zymosan-affinity of *A. mellifera's’* Vitellogenin. We hypothesized
that improving zymosan binding would enable the queen bee to bind and
transport more zymosan into the oocytes [19] thus, mediate a higher protection
of all offsprings against entomopathogenic fungi, which can be used for
treating *Varroa* mite infestation.

With this aim, we needed to decide on a suitable workflow for generating and
screening a library. We quickly discovered that surface display is one of the
most widely used and effective methods for affinity engineering. It uses the
capability of cells to produce and transport proteins onto their cell surface
where they are easily accessible for screening, circumventing laborious
purification. Single cell flow cytometry and fluorescent activated cell
sorting (FACS) downstream make it possible to screen large libraries and
isolate mutants that display proteins with the desired traits [20–24].

Multiple organisms can display mutated libraries such as phages [25, 26],
bacterial [27], insect [28], mammalian [29, 30] and yeast cells [31–33]. Out
of these options, we chose yeast as the system to use, as it offered the most
complete platform with the least disadvantages. They are easier to cultivate
and employ in experiments compared to insect or mammalian cells, while still
being a eukaryotic expression system, meaning they can incorporate
posttranslational modifications and guarantee correct protein conformation.
Exactly these properties are not offered by phages and bacteria, making yeast
the ideal organism for the screening of Vitellogenin mutant libraries.

Among yeast species, *Saccharomyces cerevisiae* is the most common
choice for the display of protein libraries [31–33]. In general, a yeast
surface display construct is composed of several elements (Fig. 1). A cell
wall protein (CWP) interacts with a polypeptide on the cell wall, which in
turn is typically linked to Glycosylphosphatidylinositol (GPI) [34, 35],
thereby anchoring the construct onto the cell surface. The protein of interest
(POI) is fused to the CWP via a flexible linker. It allows both the CWP and
POI to fold and move independently from one another, increasing the viability
of the displayed protein [36–38]. Lastly, an epitope tag is included in the
construct. The epitope-tag allows the quantification of the fusion protein
expression and the subsequent normalization of the measured binding affinity
[24, 39].

There are several CWPs available as anchors. We decided to use the widely
employed agglutinin mating protein Aga2p, which forms two covalent disulfide
bonds with Agα1p on the cell surface. Aga2p also serves as a secretion factor
for the construct, reducing the size and complexity of the expressed protein,
as there is no need for a separate factor [31, 34, 40].

We decided to use a (G4S)3 linker as it offers a good
compromise between flexibility and length. The polar amino acids also form
hydrogen bonds with the surrounding water molecules, reducing the interaction
of the linker with the protein domains [41].

The epitopes we used for quantification were the HA epitope and the Myc tag,
which are very prevalent in many antibody engineering studies. These often
have similar aims to our project in increasing binding affinity [24, 42, 43].
We used the two epitopes as a form of redundancy with the HA at the N-terminus
and the cMyc C-terminal. If steric interference would have made the cMyc tag
inaccessible for antibody binding, we could use the HA tag as a substitute.

The last decision we had to make for our construct was which parts of the
protein to display. Vitellogenin is a large protein with a size of
202 kDA. Considering there was no literature regarding displayed proteins
of this size and that full Vitellogenin secretion from yeasts was previously
shown to be unsuccessful [44], we decided to display the different
Vitellogenin domains.

![This figure shows a schematic view of the used surface display construct. It shows a yeast cell with Agα1p on the cell surface. It is connected to Aga2p through to disulfide briges. The Aga2p is connected to HA which is connected to a Vitellogenin domain. The domain is binding a fluorescent labled PAMP. At the c-terminus is a myc tag, binding a flourescent marker. . Next is the HA epitope and the Vitellogenin domain of interest. Attached to the Vitellogenin is the FTSC labeled PAMP zymosan. The cMyc tag is shown at the c terminus with antibodies attached to it, indicated by the red point.](https://static.igem.wiki/teams/4675/wiki/results/1-ysd-onstruct.png)

Figure 1: Schematic view of the used surface display construct. Aga2p is
anchored to the cell wall via two disulfide bridges formed with Agα1p. The
linker (not shown) connects to the rest of the construct. Next is the HA
epitope and the Vitellogenin domain of interest. Attached to the
Vitellogenin is the FTSC labeled PAMP zymosan. The cMyc tag is shown at the
c‑terminus with antibodies attached to it, indicated by the red point.

Analyzing the functional domains [1] (Fig. 2), we discovered, a long sequence
between the DUF1943 domain and the van Willebrand factor (vWf), which had no
proposed function. As the vWf is predicted to be primarily involved in protein
stabilization [1] we deemed this section of the protein non‑relevant to our
engineering effort. Additionally, the N-terminal β-barrel domain is a putative
transcription factor [45] linked to the protein via the cleavable poly-serin
linker [46]. Considering its role, it will not necessarily be transported into
the oocytes and likely serves no function in PAMP binding. Therefore, it was
also excluded from the surface display constructs alongside the poly-serin
linker. These decisions left us with only two domains of interest. The
alpha-helical domain which was already suggested to bind PAMPs as a sole
domain but with reduced affinity [47] and the adjacent DUF1943 domain which is
known to contribute to PAMP binding in fish and coral species [48, 49]. We
decided that reviewing the binding affinity of both domains separately, and
combined could yield us valuable insight into the mechanism of PAMP binding,
which was previously not described for
*A. mellifera's* Vitellogenin. We also decided to try a display of
a truncated Vitellogenin lacking the β-barrel domain, poly-serin linker, and
ending directly after the vWf. This resulted in four constructs to be
assembled and displayed. One displaying just the alpha-helical domain (45.5
 kDA), one containing the DUF1943 domain (34.27 kDA), a third with
the alpha-helical & DUF1943 region (97.75 kDA) and a lastly a truncated
Vitellogenin version far larger than the other domains at 144.99 kDA.

![The figure shows the simplyfied amino acid sequence of Vitellogenin and its domains. 1: Vitellogenin sequence. 2: β-barrel + polyserin linker, acts as a transcription factor linked via a cleavable polyserine linker. 3: Alphahelical, Heavily positive charged domain thought to be involved in PAMP binding. 4: DUF1943, contributes to PAMP-binding eith te alpha-helical domain. 5: vWF, suggested to be involved in proetin stabilitation](https://static.igem.wiki/teams/4675/wiki/results/2-sequence-map-neu-3x.png)

Figure 2: *Apis mellifera* Vitellogenin domains. The proposed
functions of each domain are listed below. The numbers annotate the amino
acids corresponding to the domains.

Our choice to use the Aga2p anchor gave us a clear direction for which yeast
strain to use. The widely employed *S. cerevisiae* EBY100 strain
[24, 32, 39] contains the cell wall-bound Agα1p component of the anchor
complex under the control of the galactose-inducible GAL1 promotor, making it
ideal for our concept. The vector pCTCon2 is well suited for use in this
strain, as it allows for the yeast surface display construct to also be
expressed under the GAL1 promotor and auxotrophic selection via tryptophan
dropout medium (Addgene #41843).

After constructing the display vectors, we needed to generate a highly
diverse, non-biased library. We chose error-prone PCR with the nucleotide
analogous dPTP and 8-oxo-GTP for that. It requires no prior knowledge of
domain function like saturation mutagenesis [50] and is easier to execute in a
short time frame compared to DNA shuffling [51]. The here utilized nucleotide
analogues can mediate both transitions and transversions, while introducing
less GC-bias into the mutated sequence when compared to manganese ion-based
error‑prone PCRs [52, 53].

We generated the library by amplifying the plasmid backbone, excluding the
insert, with a high-fidelity polymerase and the inserts in error-prone
reactions with varying nucleotide analogue concentrations to cover a wider
range of mutation rates. Generating libraries with the correct mutation rates
is critical when considering, that 30-50% of mutations are deleterious to the
protein function [54, 55]. This makes highly mutated libraries unusable for
directed evolution, while too little amino acid exchanges result in only a
small and less diverse variant pool. We decided to use 0.2; 2 and 20 µM
of nucleotide analogues for our library generation to cover a wide range of
mutation ranges.

80 bp long overhangs complementary to the plasmid backbone were
introduced during the error-prone PCR to enable efficient homologous
recombination of the library into the backbone inside the yeast cells [56].
The fragments were purified and concentrated via lyophilisation, pooled with
the backbone, and used for yeast transformation via electroporation. The
transformed yeasts were first grown in SD+CAA selective medium before being
induced in SG+CAA medium inducing the presentation of the generated library
(Fig. 3).

![The figure shows an overview of the directed evolution approach. A library was created through error-prone PCR. Yeast cells were transformed with mutant library and labled with fluorescent zymosan. Screening of the cells using fluorescent -activated cell sorting.](https://static.igem.wiki/teams/4675/wiki/results/3-workflow.png)

Figure 3: Schematic overview of our directed evolution approach: initially,
we created a Vitellogenin domain mutant library through error prone PCR
using pTCON2. Following this, we introduced the library into the
*Saccharomyces cerevisiae* EBY100 strain and induced the
expression and display of the Vitellogenin domain mutants. To identify and
isolate mutants with the highest affinity for the PAMP, we employed
FTSC‑labeled zymosan as the target molecule.

The library was then prepared for FACS by initially labelling the cMyc tag
using mouse anti cMyc (ThermoFisher # 910MYCL) and goat antimouse
AlexaFluor®633 (ThermoFisher # A-21126) antibodies. We then exposed
the cells to FTSC labeled zymosan in varying concentrations. Protein variants
with improved binding kinetics are more likely to retain the bound PAMP on the
cell surface, leading to positive selection in the FACS. With this we were
able to identify Vitellogenin variants with a higher affinity for zymosan,
which can improve the transgenerational immune priming of honey bees against
enteropathogenic fungi. Enabling their usage against *Varroa* mites.

### Zymosan Labeling

To enable the screening process through FACS, it was crucial to fragment and
label the zymosan with a fluorescent dye. As mentioned above, zymosan is a
large, water-insoluble polysaccharide (approx. 300 kDa), which is why we
initially subjected it to acid hydrolysis. We chose mild conditions to keep
the zymosan fragments as large as possible so that they could still be
recognized by Vitellogenin as PAMPs while remaining water-soluble. This was
accomplished by subjecting them to a solution of 0.5 M HCl at 70 °C for a
duration of 2 hours. Following acid hydrolysis, the soluble zymosan fragments
were ultracentrifuged through a 3 kDa membrane to exclude very small sugar
oligos, which were unlikely to be recognized as PAMPs (Fig. 4).

![The figure shows the workflow of the zymosan labeling. Zymosan were labeled with FTSC. Zymosan fragments are centrifuged to seperate zymosan fragments from not usable sugar oligos.](https://static.igem.wiki/teams/4675/wiki/results/4-hyrolyse-neu3x.png)

Figure 4: Workflow Description: Acid hydrolysis and ultrafiltration for
isolation of water soluble zymosan fragments. This involved incubating the
zymosan in a 0.5 M HCl solution at 70 °C for 2 hours. After acid
hydrolysis, the resulting soluble zymosan fragments underwent
ultracentrifugation through a 3 kDa membrane to exclude small sugar oligos,
which are less likely to be recognized as PAMPs.

Subsequently, the larger zymosan fragments were labeled with the fluorescent
dye fluorescein-5-thiosemicarbazide (FTSC). This dye reacts with the reducing
ends of sugars, causing them to form hydrazones, which are subsequently
reduced by sodium cyanoborohydride (NaBH3CN) to the fluorescent
zymosan derivates. For ideal labeling conditions, a molar ratio of 8:1 (FTSC
to reducing ends) and 70 µL of 0.4 M NaBH3CN per mg of
zymosan, dissolved in a mixture of 7:3 DMSO:HOAc, can be used (Fig. 5).

![The figure schows the reaction of the zymosan labeling with 5-FTSC (fluorescein-5-thiosemicarbazid).](https://static.igem.wiki/teams/4675/wiki/results/5-zymosan-labeling-1.png)

Figure 5: Reaction of the FTSC labeling: FTSC chemically reacts with the
reducing end of sugars, leading to the formation of hydrazones. These
hydrazones are then reduced into fluorescent zymosan derivatives using sodium
cyanoborohydride (NaBH3CN). For optimal labeling conditions, a
molar ratio of 8:1 (FTSC to reducing ends) and the addition of 70 µL of 0.4 M
NaBH3CN per milligram of zymosan, dissolved in a mixture of 7:3
DMSO to HOAc was used.

After that, the FTSC-labeled zymosan was again passed through a 3 kDa membrane
to remove the unreacted chemicals of the reaction. The FTSC-labeled zymosan
was then precipitated in ethanol, a solvent in which the FTSC dye is soluble,
to remove residual FTSC. To validate the successful acid hydrolysis and FTSC
labeling, various methods were employed. Firstly, the acid hydrolysis was
verified through Matrix-Assisted-Laser-Desorption-Ionization with
Time-Of-Flight analysis of released ions for mass spectrometry (MALDI TOF MS).
Further confirmation was obtained by observing fluorescence of the
precipitated zymosan under UV light as well as unlabeled zymosan as a negative
control. The protocols for this we created ourselves, as there was no such
protocol available. You can find it in
[contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D).

### Results

Acid hydrolysis was verified by MALDI TOF MS. The results are depicted in
Figure 6. Glucose has a molecular weight of 180 Da, and when forming the
glycosidic bond, water with a molecular weight of 18 Da is eliminated.
Considering that zymosan is a polymer comprised of beta-1,3- and
beta-1,6-linked glucose monomers, the peaks in the mass spectrum exhibiting a
consistent gap of 162 m/z prove that the hydrolysis was successful.
Furthermore, the data also reveals a decrease in the quantity of zymosan
fragments with increasing molecular size. As evident from the data, the
hydrolysis indeed proved effective.

![This figure shows the mass spectrometry histogram plot of zymosan fragments after acid hydrolysis. The mass spectrum displays consistent 162 m/z gaps in the peaks, highlighting the zymosan structure, composed of beta-1,3 and beta-1,6 glucose monomers.](https://static.igem.wiki/teams/4675/wiki/results/6-zymosan-labeling-2.png)

Figure 6: Mass Spectrometry histogram plot of zymosan fragments after acid
hydrolysis. Remarkably, the mass spectrum displays consistent 162 m/z
gaps in the peaks, highlighting the zymosan structure, composed of beta-1,3
and beta-1,6 glucose monomers.

Following the successful fragmentation of the zymosan polymer, we proceeded to
label the resulting fragments using the fluorescent dye FTSC. The labeled
polymer demonstrated pronounced fluorescence at 492 nm, as depicted in
figure 7. Following the washing steps, the flowthrough also exhibited
fluorescence. This fluorescence decreased with each subsequent wash step,
indicating a successful removal of the residual FTSC. Conversely, the negative
control, consisting of unlabeled zymosan fragments, did not exhibit any
fluorescence signal.

![This figure shows the fluorescence at 492 nm at A: labeled and purified zymosan fragments, B: flowthrough of washing step 1, C: flowthrough of washing step 2, D: flowtrough of washing step 5, E: flowthrough of washing step 4, F) unlabeled zymosan fragments as negative control.](https://static.igem.wiki/teams/4675/wiki/results/7-zymosan-labeling-3.png)

Figure 7: Fluorescence at 492 nm. From left to right: Labeled and
purified zymosan fragments flowthrough of washing step 1 flowthrough of
washing step 2 flowthrough of washing step 3 flowthrough of washing step 4
unlabeled zymosan fragments as negative control

### Generation of the Base Construct

First, we constructed the base surface display plasmid via Gibbson Assembly.
It contained the functional elements of the surface display, but not the
Vitellogenin domains. We ran colony PCRs, amplifying the complete insert, to
verify the presence of positive clones. The expected fragment size was
374 bp, which we could observe for several colonies. We send several
colonies that showed the right-sized band to sequencing where the correct
assembly of the construct was confirmed (Fig. 8).

### Constructs

![This figure shows a gel electrophoresis of a colony PCR of the pCTCon2 vector containing the yeast surface display functional units is shown. Several colonies show bands of the expected size of 374 bp. The used marker was a 1 kb plus ladder.](https://static.igem.wiki/teams/4675/wiki/results/8-gelbild-i-1.png)
![This figure shows the sequence of the colony, confirming the correct insertion of the fragment.](https://static.igem.wiki/teams/4675/wiki/results/9-vgfull-ysd-construct-map.png)

Figure 8: In the picture on top, the gel electrophoresis of a colony PCR of
the pCTCon2 vector containing the yeast surface display functional units is
shown. Several colonies show bands of the expected size of 374 bp. The
used marker was a 1 kb plus ladder. The bottom picture shows the sequence
of the colony, confirming the correct insertion of the fragment.

Next, we needed to integrate the four different Vitellogenin fragments into
the construct. We amplified the domains out of a fully assembled and codon
optimized Vitellogenin sequence, ordered from IDT. After transformation we ran
colony PCRs again, before sending the constructs to be sequenced to verify
their identity.

#### Vg\_α‑helical

![This figure shows the schematic of Apis mellifera Vitellogenin. The α helical domain chosen for display is highlighted.](https://static.igem.wiki/teams/4675/wiki/results/10-alphahelical-neu-3x.png)

Figure 9: Schematic of *Apis mellifera* Vitellogenin. The
α‑helical domain chosen for display is highlighted.

Two colonies of Vg\_α‑helical showed the expected band at ~1400 bp. Both were
sent to sequencing, where one was confirmed positive (Fig. 10).

![This upper figure shows a gel electrophoresis of a colony PCR of the pCTCon2_Vg_α‑helical on the left. The first band shows the expected size of ~1400 bp while the right band runs to high. The right side shows the sequence result of the colony, confirming it to contain the Vg_α‑helical insert. ](https://static.igem.wiki/teams/4675/wiki/results/11-gelbild-ii.png)
![ The lower figure shows the sequence result of the colony, confirming it to contain the Vg_α‑helical insert.](https://static.igem.wiki/teams/4675/wiki/results/12-vgalpha-ysd-construct-map.png)

Figure 10: At the top the gel electrophoresis of a colony PCR of the
pCTCon2\_Vg\_α‑helical is shown. In the left pocket, a 1 kb plus ladder was
run. By comparison, the middle band shows the expected size of ~1400 bp
while the most right band runs to high. The bottom figure shows the sequence
result of the colony, confirming it to contain the Vg\_α‑helical insert.

#### Vg\_DUF1943

![This figure shows a schematic of Apis mellifera Vitellogenin. The DUF1943 domain chosen for display is highlighted.](https://static.igem.wiki/teams/4675/wiki/results/12b-duf-neu-3x.png)

Figure 11: Schematic of *Apis mellifera* Vitellogenin. The DUF1943
domain chosen for display is highlighted.

Two colonies showed the expected band at ~1000 bp. Both were sent for
sequencing, and both were confirmed to contain the correct insert (Fig. 12
only one shown).

![The upper figure shows a gel electrophoresis of three colony PCRs of the pCTCon2_Vg_DUF1943 in comparison to a 1 kb plus ladder. The two colonies on the left show the expected band size of ~1000 bp. The right side of the figure shows the sequence result of one colony, confirming it to contain the Vg_α-DUF1943 insert](https://static.igem.wiki/teams/4675/wiki/results/13-gelbild-iii.png)
![The lower figure shows the sequence result of one colony, confirming it to contain the Vg_α-DUF1943 insert](https://static.igem.wiki/teams/4675/wiki/results/15ysd-duf1943-construct-assembled-new-map.png)

Figure 12: At the top the gel electrophoresis of three colony PCRs of the
pCTCon2\_Vg\_DUF1943 in comparison to a 1 kb plus ladder is shown. The two
colonies on the left show the expected band size of ~1000 bp. The bottom
figure shows the sequence result of one colony, confirming it to contain the
Vg\_α-DUF1943 insert.

#### Vg\_α‑helical+DUF1943

![This figure shows a schematic of Apis mellifera Vitellogenin. The α‑helical+DUF1943 domain chosen for display is highlighted.](https://static.igem.wiki/teams/4675/wiki/results/16-alphaduf-neu-3x.png)

Figure 13: Schematic of *Apis mellifera* Vitellogenin. The
α‑helical+DUF1943 domain chosen for display is highlighted.

Again, two colonies showed the expected band at ~2200 bp. Both were sent to
sequencing where they were confirmed to contain the correct construct (Fig. 14
only one sequence shown).

![The upper figure shows a gel electrophoresis of five colonies PCR of the pCTCon2_Vg_α‑helical+DUF1943 in comparison to a 1 kb plus ladder. Two colonies show the expected band size of ~2200 bp. The right side of the figure shows the sequence result of one colony, confirming it to contain the Vg_α‑helical+DUF1943 insert.](https://static.igem.wiki/teams/4675/wiki/results/17-gelbild-iv.png)
![The lower figure shows the sequence result of one colony, confirming it to contain the Vg_α‑helical+DUF1943 insert. ](https://static.igem.wiki/teams/4675/wiki/results/18-ysd-alphahelical-duff-construct-assembled-new-map.png)

Figure 14: At the top the gel electrophoresis of five colonies PCR of the
pCTCon2\_Vg\_α‑helical+DUF1943 in comparison to a 1 kb plus ladder is
shown. Two colonies (third lane, sixth lane) show the expected band size of
~2200 bp. The bottom figure shows the sequence result of one colony,
confirming it to contain the Vg\_α‑helical+DUF1943 insert.

#### Vg\_truncated

![This figure shows a schematic of Apis mellifera Vitellogenin. The truncated domain chosen for display is highlighted. ](https://static.igem.wiki/teams/4675/wiki/results/19-full-neu-3x.png)

Figure 15: Schematic of *Apis mellifera* Vitellogenin. The truncated
domain chosen for display is highlighted.

One colony showed a promising fragment at ~3900 bp. It was sent to sequencing
where it was confirmed to contain the correct fragment (Fig. 16).

![The upper figure showas a gel electrophoresis of a colony PCR of the pCTCon2_Vg_truncated in comparison to a 1 kb plus ladder. The band shows the expected size of ~3900 bp. The right side of the figure shows the sequence result of the colony, confirming it to contain the Vg_truncated insert. ](https://static.igem.wiki/teams/4675/wiki/results/20-gelbild-v.png)
![The lower figure shows the sequence result of the colony, confirming it to contain the Vg_truncated insert.](https://static.igem.wiki/teams/4675/wiki/results/21-ysd-vgtruncated-construct-assembled-map.png)

Figure 16: At the top the gel electrophoresis of a colony PCR of the
pCTCon2\_Vg\_truncated in comparison to a 1 kb plus ladder is shown. The
band shows the expected size of ~3900 bp. The bottom figure shows the
sequence result of the colony, confirming it to contain the Vg\_truncated
insert.

### Verification of Protein Display

The first step of our engineering was to verify the correct display of our
constructs. We were particularly concerned about the display of our larger
constructs, Vg\_alpha+DUF and Vg\_truncated.

We used eight different controls (Fig. 17) to compare our constructs with a
cell control, single antibody controls with the first and secondary
antibodies, and a double antibody control. Each control was also run after
incubation with zymosan to check for background yeast interaction with the
PAMP.

![This figure shows a list of controls used to verify the correct display of Vitellogenin domains. 1: Yeast cell 2: Yeast cell + labeled zymosan 3: yeast cell+ primary antibody 4: yeast cell + primary antibody + labeled zymosan 5: yeast cell + secondary antibody 6: yeast cell + secondary antibody + labeled zymosan 7: yeast cell + primary antibody + secondary antibody 8: yeast cell + primary antibody + secondary antibody + labeled zymosan](https://static.igem.wiki/teams/4675/wiki/results/22-ysd-controls.png)

Figure 17: Controls used to verify the correct display of the Vitellogenin
domains.

Before analyzing flow cytometry data, it is important to isolate the so-called
“events” relevant to the experiment. An event is any signal measured by a flow
cytometer without any differentiation of its source. This means that events
originating from debris, dead cells, or other sources need to be excluded
before continuing with the analysis. There are several different measurements
used to determine which accumulation of events, or “population,” shows the
desired cells. Front scatter (FSC) measures the light passing through the
event, while side scatter (SSC) measures the light being refracted sideways.
The signal of each can be subdivided into a height (e. g., FSC-H), width
(e.g., FSC-W), and area (e.g., FSC-A value). Height measures the signal
intensity, while width measures the time the event passes through the beam.
The integral of the measured event generates the area value. Different
combinations of these can be used to investigate the properties of the
measured events.

FSC-A vs SSC-A is used to isolate the cells from debris or other impurities.
FSC-A indicates cell size, while SSC-A shows the internal complexity, also
called granularity. As seen in Fig. 18, we observed a well-defined cell
population with no debris and other populations that would indicate dead cells
or other cell types. An area, or ‘gate,’ was set marking the cells in it for
further analysis. We then checked if our sample contained cell aggregations.
Cells can either stick together, or can by chance, be measured as a single
event. As the presence of such multicell events would interfere with data
interpretation they need to be sorted out. We did this via FSC-A vs FSC-H
plots. Cell aggregations would appear as a lower population because of their
different FSC-A composition. They have a larger FSC-W value due to their size
with a lower FSC-H, which they can be sorted by. No subpopulations were
observed in FSC‑A/FSC‑H, indicating that only single cells were measured with
no cell aggregations present.

![This figure shows a graphic for the cell control for both antibodies.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/1-1.png)

A

![This figure shows a graphic for the cell control for both antibodies.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/2-1.png)

B

Figure 18: A) Front scatter area (FSC-A) vs side scatter area (SSC-A) plot of
the cell control + double antibodies + 0.2 mg of zymosan is shown. One
distinct population is visible in this plot, indicating that no other cell
morphologies or dead cells are present, as well as no other debris. B) Front
scatter area (FSC-A) vs front scatter height (FSC-H) plot. The presence of
only one distinct population indicates that no cell aggregations are present
in the sample, as these would appear lower on the plot.

We then investigated which constructs presented the display construct. We used
the two fluorescent markers fluorescein-5-thiosemicarbazide (FTSC) attached to
zymosan and AlexaFluor®633 as our secondary antibody respectively
to measure the presence of the PAMP in proportion to the displayed protein. A
cell control with both antibodies and 0.2 mg of zymosan served as the
baseline, which the displays were compared to. A gate named “high display” was
set (Fig. 19) covering the whole PAMP fluorescence range, with the left border
of the antibody fluorescence range at the edge of the main population. This
gate included all cells that showed interaction with the antibodies and
excluded those that did not. The event count in this gate was subsequently
analyzed and the constructs were compared to the cell control. A higher
percentage of cells in this gate indicates higher binding of the secondary
antibody, which is expected if our construct is displayed.

As seen in Fig. 19 the displays of the Vg\_α‑helical, Vg\_α‑helical+DUF1943, and
Vg\_truncated domains were successful. The cell control shows a small
population of cells interacting with the antibody, leading to 8.75% of the
events being in the set gate. We tried normalizing the measurements with the
control, but the low signal-to-noise ratio of our samples led all measured
signals to be quashed to zero, making them unusable. But even without
normalization we could observe a clear increase in event count across the
three constructs. As expected, the size of the domains had a great impact on
display efficiency with the Vg\_α‑helical domain showing the strongest display,
at 11.75% more events inside the chosen gate, while the larger
Vg\_α‑helical+DUF1943 was displayed to a slightly lower degree with an 10.05%
increase in events. To our delight, we were also able to observe the display
of the 150 kDA truncated sequence at a sufficient level, at 4.65% over
the cell control. Only the separate display of the Vg\_DUF1943 domain could not
be demonstrated as the FACS runs were unusable due to technical issues of the
cytometer.

![This figure shows graphics for different Vitellogenin domains.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/3-1.png)

A

![This figure shows graphics for different Vitellogenin domains.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/4-1.png)

B

![This figure shows graphics for different Vitellogenin domains.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/5-1.png)

C

![](https://static.igem.wiki/teams/4675/wiki/results/flowjo/6-1.png)

D

Figure 19: The plots shown compare AlexaFluor®633 fluorescence of
the secondary antibody (DyLight633 channel) to the FTSC fluorescence of
Zymosan (AlexaFluor®488 channel) of the A) double antibody cell
control + 0.2 mg/mL zymosan, B) Vg\_α‑helical + 0.2 mg/mL zymosan, C) Vg\_
α‑helical+DUF1943 + 0.2 mg/mL zymosan and D) Vg\_truncated + 0.2 mg/mL
displays. Higher fluorescence of AlexaFluor®633 corresponds to
higher protein display while higher FTSC fluorescence indicates higher zymosan
binding. The gate ´high display´ was set at the right edge of the cell
controls main population. Vg\_α‑helical (B) shows the strongest secondary
antibody fluorescence with 20.5% in the ´high display´ gate.
Vg\_α‑helical+DUF1943 (C) and Vg\_truncated (D) show lower display efficiencies
at 18.8% and 13.4% respectively. Only the cells in the high display gate were
used for further affinity analysis.

#### Vg\_DUF1943 Display Analysis

Before analysis we viewed every sample in SSC-A against time plot to see if
any irregularities were present. Almost all runs showed perfect readings but
the VG\_DUF1943 run had severe dropouts and shifts in readings (Fig. 20). We
tried to negate these problems via a more extensive gating protocol. We first
used a gate called ´FACS failure´ to remove all events from the measurement
that fell into the timeframe of the dropout. Unfortunately, when plotting
these cells using FSC-A against SSC-A we still saw a very irregular event
pattern. One diffuse population was visible at a similar position to the
normal cells, with a second population at SSC-A approaching zero. We used a
gate to isolate the events at the usual position for cells and proceeded to
analyze these. When gating them with the same ´high display´ and ´high
binding´ gates as other samples, we realized that they possessed a display
value about half of the cell control and only showed baseline PAMP binding
affinity. We were unable to run another measurement due to time constraints
and were therefore unable to make a verdict on the display of the Vg\_DUF1943
domain.

![This figure shows graphics for different Vitellogenin domains.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/7-1.png)

A

![This figure shows graphics for different Vitellogenin domains.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/8-1.png)

B

![This figure shows graphics for different Vitellogenin domains.](https://static.igem.wiki/teams/4675/wiki/results/flowjo/9-1.png)

C

![](https://static.igem.wiki/teams/4675/wiki/results/flowjo/10-1.png)

D

Figure 20: Irregular FACS run of VG\_DUF1943. A) A long signal dropout is
visible in the SSC‑A/time plot which was eliminated via the gate ´FACS
failure´. B) The following FSC‑A/SSC‑A plot showed a very irregular event
pattern with one diffuse cell population and a second population at a very low
SSC‑A. The cell population was used and analysed as previously using the C)
´high display´ and D) ´high binding´ gates where the cells showed antibody
binding below the level of the cell control and only baseline PAMP affinity.

#### Measurement of Domain PAMP Affinity

After verifying the correct display of our domains of interest we investigated
which of them were involved in Vitellogenin’s PAMP binding affinity. A new
gate was created in the “high display” gate called ´high binding´ (Fig. 21).
It was set to include the top ~50% events of the highest binding domain. This
range was chosen as it reduced the effect of both positive and negative
outliers. A gate set too high would increase the share of positive outliers in
the overall events, while a broader gate, more focused on lower binding
domains, would include almost all events of strong binding domains, making
these domains more difficult to characterize.

The cell control again showed some affinity which could not be compensated. We
expected the Vg\_αhelical domain to have decent binding affinity, as it was
previously suggested to be the main factor for PAMP recognition [57], but we
could not confirm this. With 19.2% of events in the set gate it was only
marginally more than the cell control. We suspected potential misfolding to be
responsible for its poor binding characteristics,
[but *in silico* analysis did not support this](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D). The Vg\_alpha+DUF1943 domain showed the strongest binding, with 52.7% inside
its gate, strongly suggesting a role of the Vg\_DUF1943 domain in PAMP binding.
This was further supported by the measurements of Vg\_truncated. It had just
35.3% of events in the ´high binding´ gate, indicating that the remaining
protein does not, or only to a small degree, participate in zymosan PAMP
recognition.

As we had no ability to investigate the Vg\_DUF1943 affinity we were unable to
determine if zymosan binding is only mediated by the Vg\_DUF1943 domain, or if
the Vg\_α‑helical contributes to it as well. Based on earlier studies
describing Vg\_α‑helical PAMP recognition and the marginal binding affinity
observed in our measurement we suggest that both domains are cooperatively
binding the polymer.

![](https://static.igem.wiki/teams/4675/wiki/results/flowjo/11-1.png)

A

![](https://static.igem.wiki/teams/4675/wiki/results/flowjo/12-1.png)

B

![](https://static.igem.wiki/teams/4675/wiki/results/flowjo/13-1.png)

C

![](https://static.igem.wiki/teams/4675/wiki/results/flowjo/14-1.png)

D

Figure 21: The plots shown compare AlexaFluor®633 fluorescence of
the secondary antibody (DyLight633 channel) to the FTSC fluorescence of
Zymosan (AlexaFluor®488 channel) in the ´high display´ gate of the
A) double antibody cell control + 200 µg/mL zymosan, B)
Vg\_α‑helical + 200  µg/mL zymosan, C) Vg\_α‑helical+DUF1943
+ 200 µg/mL zymosan and D) Vg\_truncated + 200 µg/mL. Higher
fluorescence of AlexaFluor®633 corresponds to higher protein
display while higher FTSC fluorescence indicates higher zymosan binding. The
gate ´high binding´ was set to include half the events of the strongest
binding domain. This guaranteed that the focus on outliers of both strong and
weak binding domains was minimized and that lower binding domains were
included in the measurement to a sufficient degree. Vg\_ αhelical+DUF1943 (C)
showed the strongest binding with 52.7% of events inside the gate, binding
stronger than Vg\_truncated (D) at 35.3% and much stronger than Vg\_ αhelical
(B) at 19.2% which was previously suggested for PAMP affinity.

Based on these results, we chose Vg\_alpha+DUF1943 for further work. It
presented the best balance between display efficiency and strong binding
affinity. Before screening the library, we needed to determine the binding
kinetics of Vg\_alpha+DUF1943. Without the correct PAMP concentration, we could
either saturate the different protein variants regardless of their affinity or
present the variants with too little PAMP to generate a sufficient signal to
sort the cells. We measured a range of zymosan dilutions between
0.02 µg/mL and 200 µg/mL with the display and gated them as detailed
above. We then used the event ratio to determine the degree of PAMP saturation
of the presented domains. When fitting a curve to our data points assuming
Michaelis‑Menten-like kinetics we calculated a KD = 0.61 µg/mL. As
there is no substrate turnover with Vitellogenin KD is equal to
KM (Fig. 22). Molar concentrations could not be determined, as we
were unsure about the ratio of different zymosan oligomers in the labeled
solution. Based on this we chose a concentration of 2 µg/mL for library
screening as it presented a concentration which should saturate cells to ~75%.
We expected that this would improve library sorting as binding cells would be
strongly labeled and easier to isolate. Lower zymosan concentrations around
KD or below would be more sensitive but could compromise sorting as
binding cells are harder to differentiate from low or non‑binding ones.

![This figure shows a curve of the zymosan concentration in µg/mL against the Vitellogenin zymosan saturation. The curve is exponentiel until around (10|0.9), then it runs against 1.](https://static.igem.wiki/teams/4675/wiki/results/37-vg-zymosan-fit.svg)

Figure 22: Kinetics fit to the binding affinity of Vg\_α‑helical+DUF1943
surface display. Different zymosan concentrations between 0.2 µg/mL and
200 µg/mL were measured and normalized using the cell control. Based on this
kinetics fit, the KD was determined to be 0.61 µg/ml.

We generated the library via error‑prone PCR using 0.1, 1, and 10 µM of
8‑o‑xoGTP and dPTP each. The fragments and a backbone amplified by a
high-fidelity polymerase were used for the transformation of yeast.
Subsequently, the cells were prepared for sorting. We decided to use two gates
for sorting. One gate isolated cells with the combined properties of a high
display with high binding affinity, while the other was set to sort out cells
with slightly lower display and strong binding. These were chosen to not favor
cells displaying variants with improved display characteristics, as these
would be enriched in a high display gate. Due to time constraints, we were
limited to a single FACS run and had to sort single cells immediately.
Iterative FACS runs would have enabled us to sort out the top percent of
binding cells and regrow them to a higher quality library, which would be
sorted again. This would enrich improved variants to a point where they could
be easily isolated with a lower risk of losing improved variants from a too
aggressive sorting approach.

Unfortunately, when analyzing the data of the cell sorting it became apparent
that the binding affinity of all libraries was not significantly different
from the level of the cell control (Fig. 23).

![This figure shows a curve of zymosan concentration against the percentage of cells in the high display gate.](https://static.igem.wiki/teams/4675/wiki/results/38-vergleich-library-gegen-negativkontrolle.svg)

Figure 23: Binding affinities of the Vg\_α‑helical+DUF1943 libraries which were
generated using 0.2, 2 and 20 µM of nucleotide analogues. All libraries
were measured using 2 µg/mL and 20 µg/mL. The cell control also
included a measurement at 10 µg/mL. The high display gate was set to include
about half of the strongest binding variants. As evident no library showed
increased PAMP affinity over the cell control which would indicate display of
functional protein.

To investigate the source of our findings, we cloned fraction of each library
into a pBSK vector and send them for sequencing. It was revealed that the
error-prone PCR amplified a range of unspecific and gapped vector fragments
(Fig. 24). This, of course, made the library unusable as the lack of the
homologous ends, only present to the left and right border of
Vg\_α‑helical+DUF1943, made recombination of the fragments inside the yeast
impossible. The reason for the poor performance of the primers was likely in
the switch from the NEB Q5® polymerase, where they were very
reliable, to a Taq polymerase required for error-prone PCR and the
accompanying changes in annealing temperature. The new reaction conditions
apparently led to unspecific primer annealing. In the PCR, smaller fragments
are generated with a higher amplification efficiency, which leads to them
enriching in the reaction. Thereby, the desired sequence is largely replaced.

![This figure shows the alignment of the fragments amplified via error-prone PCR](https://static.igem.wiki/teams/4675/wiki/results/39-ysd-alphahelical-duff-construct-assembled-new-map.png)

Figure 24: Sequences of fragments amplified via error-prone PCR. The fragments
were cloned into pBSK via blunt-end ligation and send for sequencing. It is
evident, that the primers did not bind specifically, with only one fragment
aligning to the intended Vg\_αhelical+DUF1943. The frequent occurrence of gaps
is also a severe problem for library generation.

### Outlook

While our assay proved to be a reliable and easy-to‑perform, there are some
adjustments that can be made to improve it for future applications. We would
advise the addition of a terminator like PGK1 or CYC1 in the display
constructs. It could increase protein yield and display capability of the
cells by terminating transcription directly after the protein, reducing the
metabolic burden of expression [58].

Another issue was the poor separation of displaying cells from non‑displaying
ones in the cell staining. This appears to be a potential issue for large or
flexible display constructs [39, 59]. The first approach for improving cell
staining would be to utilize the second immune epitope to avoid potential
steric hinderance of antibody binding at the c‑Myc tag. If this yields no
improvement changes in the staining protocol could prove promising. Increased
antibody incubation time or decreasing the incubation temperature present
viable approaches [39]. Wen et al. (2010) also encountered poor staining
separation issues similar to ours, which they resolved by using a single
antibody stain instead of the standard two-step antibody protocol. We think
these adjustments would greatly enhance the precision of the analysis and cell
sorting which leads to an improved identification of the displayed cells while
the carryover of non‑displaying cells is greatly improved.

For library generation we advise to check the error-prone PCR on a gel to see
if unspecific fragments are present. We did not find sources suggesting that
nucleotide analogues are responsible for unspecific primer binding or
generation of gapped PCR sequences. If our issues reoccur with adjusted
annealing temperature and primers it would be advisable to change the
error-prone PCR to a variant employing manganese ions and adjusted dNTP mixes
to counteract GC-bias.

---

## Expression of *Helicoverpa armigera* C-type Lectin 14

As an alternative approach to increase pathogen associated molecular pattern
(PAMP) binding affinity, we wanted to express the C-type lectin 14 (CTL14) of
*Helicoverpa armigera* in *Escherichia coli* for
testing in the zymosan binding assay. C-type lectins are a broad superfamily
of proteins characterized by their C-type lectin domain (CTLD). A common
feature of CTLs is calcium dependent binding of ligands [56]. These proteins
play important roles in immunity. For example, they recognize microbes, such
as fungi and bacteria, through PAMPs through various glycan structures [56].
CTLs are key factors in the immune system of invertebrates and most
importantly for insects, such as *Apis mellifera* [57, 58]. We found
out that the CTL14 of *H. armigera* can bind the PAMP zymosan [59].
Zymosan is also a major cell wall component of
*Beauveria bassiana*, one of the entomopathogenic fungi which
could be used to fight against the *Varroa* mite [60], thus making the
CTL14 a suitable protein to test in the zymosan binding assay. Furthermore, we
wanted to test a fusion protein of *Apis mellifera* Vitellogenin and
CTL14. We hypothesized that a fusion protein would show improved zymosan
binding activity, due to the binding capabilities of CTL14 and Vitellogenin.
We started planning our lab work and looking into possible expression vectors.
We identified the pET28b as a suitable expression vector for the expression of
recombinant proteins. The pET28b has the advantage that expression can be
induced through the lac-operon with isopropyl‑beta‑D‑thiogalactopyranoside
(IPTG). Additionally, we needed a possible linker for the
*A. mellifera* Vitellogenin and *H. armigera* CTL14 to
construct a fusion protein. Our research was focused on possible linkers,
which would keep the activity of both proteins intact and their structures
distinct. We simulated the protein conformation of the fusion protein of
Vitellogenin and CTL14 with AlphaFold using different linkers. In the end, we
chose to express the fusion protein with a helical linker with the amino acid
sequence A(EAAAK)2A. This linker was chosen as it was simulated to
keep the proteins structures distinct and the His-Tag accessible for
purification. More information regarding the fusion protein modeling is
available on our
[modeling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D). For the construction of the vector, we chose the
*E. coli* Top10 strain for its high transformation rates and for
the expression of the proteins we chose *E. coli* BL21, as it has
reduced protein degrading and the T7 RNA-polymerase of the T7 phage under the
control of a lac operon, making it ideal for high protein expression using the
pET28b vector. For this project, we chose *E. coli* strains
instead of *Komagataella phaffii,* as we did for the expression solely
of the *A. mellifera* Vitellogenin. *E. coli* is often
used in the literature for the expression of CTL´s [61]. Consequently, we
aimed to investigate whether the interaction between Vitellogenin and CTL14
could lead to a substantial change in binding affinity and potentially enhance
the bee's immune response positively. Thus, we opted to evaluate this
combination using a fusion protein.

Having decided on the expression strain, linker, and vector for the
expression, we started the construction of the pET28b-vector containing the
CTL14, Vitellogenin and them linked together, so three different vectors. The
proteins CTL14 and Vitellogenin were cloned solely into the pET28b vector to
normalize the binding of zymosan by them. We decided to do Gibson assemblies
for the construction of the expression vectors as the pET28b lacks the
necessary restriction sites for golden gate assemblies.

#### {% include 'animations/arrow.html' %} pET28b vector graphics

![](https://static.igem.wiki/teams/4675/wiki/results/ctl14/6-1.png)

Figure 25: pET28b\_CTL14 vector map. Incorporated in the PET28b backbone was
the Helicoverpa armigera ctl14 gene. Method used for the assembly of
the expression vector was Gibson Assembly. BsaI restriction sites flanking
the ctl14 gene were added for iGEM compatibility. A C-terminal 10x his-tag
was added to the ctl14 for purification of the recombinant protein. For the
expression the *Escherichia coli* BL21 strain was to be used.

![](https://static.igem.wiki/teams/4675/wiki/results/ctl14/7-1.png)

Figure 26: pET28b\_Vg vector map. Incorporated into the pET28b backbone was
the *Apis mellifera* vitellogenin gene. Method used for the
assembly of the expression vector was Gibson Assembly. BsaI restriction
sites flanking the vitellogenin were added for iGEM compatibility. A
C-terminal 10x his-tag was added to the vitellogenin for purification of the
recombinant protein. For the expression of the Vitellogenin
*Escherichia coli* BL21 was to be used.

![](https://static.igem.wiki/teams/4675/wiki/results/ctl14/9.png)

Figure 27: pET28b\_Vg-linker-CTL14 vector map. Incorporated into the pET28b
backbone was the linker protein of *Apis mellifera* Vitellogenin
and Helicoverpa armigera CTL14. The Vitellogenin and CTL14 were linked
through a helical linker with the sequence A(EAAAK)2A. BsaI
restriction sites flanking the linker protein were added for iGEM
compatibility. The method used for the construction of the expression vector
was Gibson Assembly. A C-terminal 10x his-tag was added to the ctl14 for
purification of the recombinant linker protein. For the expression of the
linker protein Escherichia coli BL21 was to be used.

Our plan was to add BsaI restriction sites flanking the genes during the
construction of the expression vectors for RCF[1000]compatibility. The protein
expression of the three recombinant proteins would then have been induced
through the lac-operon located on the pET28b vector.

We began by preparing the *vitellogenin* and *ctl14* genes,
cloning a 10x His-tag onto the C-terminal ends of the genes by PCR
amplification. This would allow us to purify the proteins later through
affinity chromatography. Gel electrophoresis was used to verify the length of
the DNA fragments and is shown in Fig. 28.

![all relevant information in caption](https://static.igem.wiki/teams/4675/wiki/results/ctl14/1.png)

Figure 28: Agarose gel of the PCR, which added the His10 tag to the
Vitellogenin and CTL14 gene. The used ladder is the 1 kb Plus ladder from
New England Biolabs. Nr.1 shows the Vitellogenin PCR and Nr.2 shows the CTL14
PCR. Both show the desired length of around 5,4 kb for Vitellogenin and
1 kb for CTL14.

![all relevant information in caption](https://static.igem.wiki/teams/4675/wiki/results/ctl14/2.png)

Figure 29: Agarose gel of the linearization of the pET28b vector. The used
ladder is the 1 kb Plus ladder from New England Biolabs. Lane 1 shows
the linearized vector at around 5,5 kb.

In the next step, the previously amplified fragments were appended to the
pET28b vector using PCR with optimized primers to create the necessary
overhangs for Gibson Assembly (Fig. 29). Initially, primers were used to
create an overhang that included specific bases from the pET28b vector,
ensuring that the fusion sides for the Gibson Assembly were located in the
pET28b vector and not in the genes. For this purpose, the pET28b empty vector
was linearized at the desired locations by PCR, with the ends containing the
necessary overhangs for Gibson Assembly.

Following the amplification, a DpnI digestion was done to remove the backbone
from the reaction mixture and increase the cloning efficiency. DpnI is a
restriction enzyme cutting only methylated DNA, which is only present in the
plasmid isolated from *E. coli* and not the PCR product.
Subsequently, two Gibson assemblies were performed for the linearized vectors
pET28b\_Vg and pET28b\_CTL14, followed by a transformation into
*E. coli* Top10. Since the pET28b vector contains an antibiotic
resistance gene for chloramphenicol, selection was performed on LB agar plates
supplemented with chloramphenicol. The grown colonies were checked for the
presence of the desired genes using a colony PCR. No positive colonies were
found during screening.

![all relevant information in caption](https://static.igem.wiki/teams/4675/wiki/results/ctl14/3-1.png)

Figure 30: Agarose gel of the pET28b vector linearized with the new primers
for Vitellogenin. The used ladder is the 1 kb Plus ladder from New
England Biolabs. Lane 1 shows the linearized pET28b\_Vg vector after DpnI
Digestion at around 5,5 kb.

In consultation with our instructors, we decided to subject the pET28b vector
to extended DpnI digestion overnight. Additionally, we optimized the Gibson
assembly overhang primers to not only extend the overhang in the pET28b vector
but also extend the vector into the genes. These overhangs were designed to be
20 to 44 bases in length. Unfortunately, the results with the extended Gibson
assembly overhang primers were unsatisfactory, prompting us to attempt
gradient PCR and touchdown PCR to create optimal conditions for the primers.
After many optimization steps, we were able to successfully complete the PCRs
and amplify the genes with the necessary overhangs we needed for cloning them
into the pET28b vector. One result is shown in Fig. 30.

At this point, we shifted our focus on the project involving a vector in which
Vitellogenin is attached to CTL14 using a protein linker. A His-tag was first
cloned onto the C-terminal end of the *CTL14*
gene through PCR. Subsequently, optimized primers were used again to clone the
protein linker onto the C‑terminal end of the
*vitellogenin* gene. Due to the linker between the Vitellogenin and
CTL14, different Gibson overhangs were cloned via PCR. We used the same
linearized vector from the pET28b\_Vg plasmid for the Gibson Assembly. The
Gibson Assembly for the pET28b\_Vg\_alphahelical\_CTL14His10xC was then carried
out and transformed into *E. coli* Top10. Unfortunately, no
positive colonies were observed in subsequent colony PCRs. We repeated the
colony PCRs using optimized primers for the incorporated genes, but still, no
positive colonies could be detected.

![The picture shows the four steps in which the CTL14 project workflow was   split. The first step was gathering information and therefore discovering the   CTL14 protein of <em>Helicoverpa armigera a</em>nd a useful expression vector.   The second step was the designing of all the laboratory work. The biggest part   was the simulations of suitable linkers for the fusion protein. In the third   step the designed vectors were constructed and controled. The last step   contains the expression of the fusion protein in <em>E. coli</em> BL21.](https://static.igem.wiki/teams/4675/wiki/results/ctl14/4-neu.png)

Figure 31: Workflow for the expression of the
*Apis mellifera* Vitellogenin and
*Helicoverpa armigera* CTL14 linker protein.

Despite encountering similar problems, we diligently attempted to troubleshoot
and rectify the issues. However, we regret to report that we were not
successful in assembling the desired vector, and further investigation was
impeded by time constraints. Therefore, we reached a point where we were
unable to explore additional potential sources of error due to the limited
available time for our research.

---

## Expression of *Apis mellifera* Vitellogenin

![The figure shows a flow chart of the workflow for the expression of Vitellogenin. 1. gathering information, 2. planning the lab work, 3. contructing the vectors, 4. expression in Komagataella phaffii](https://static.igem.wiki/teams/4675/wiki/results/evvg/1-worklfow-evvg-3x.png)

Figure 32: Workflow for the expression of
*Apis mellifera* Vitellogenin in *Komagataella phaffii* CBS7435
PEP4 KO.

The aim of our project was to express the optimized
*Apis mellifera* Vitellogenin, which was created either through
directed evolution or modeled in the dry lab via rational design choices, for
further analysis and characterization. The expression system used was the
yeast *Komagataella phaffii,* formerly *Pichia pastoris.*

Our research into the *A. mellifera* Vitellogenin began after we
had decided to tackle the problem V*arroa* mites and other pests pose
to Western honey bees. We became aware that Vitellogenin is glycosylated under
native conditions, thus directing our research towards an organism capable of
glycosylation [1]. As a eukaryote, *Komagataella phaffii* is capable of
glycosylation and is often used for the expression of glycosylated proteins
instead of *Escherichia coli,* as glycosylation does not occur in
the latter [62–66]. For this reason, we chose *K. phaffii* as the
organism for expression instead of *E. coli*. We acquired the
*K. phaffii* CBS7435 PEP4 KO strain in cooperation with the Institute
for Microbiology and Biotechnology from the University of Vienna. The strain
exhibits reduced protein degradation, as the knocked-out *pep4* gene is
the precursor of a major protease.

![The figure shows the <em>Saccharomyces cerevisiae</em> shuttle vector pSB3CY_His. The plasmid contains the <em>S. cerevisiae</em> origin of replication 2µ. The plasmids contains the a selction marker for histidine.](https://static.igem.wiki/teams/4675/wiki/results/evvg/2-psb3cy-his.png)

Figure 33: pSB3CY\_His ([BBa\_K4188002](http://parts.igem.org/Part%3ABBa_K4188002)) plasmid map. This level two shuttle vector, also called pSB3CY\_His, was
designed by the previous iGEM Münster 2022 team. BBa\_K4188002 was used as
the backbone during the construction of the expression vectors and the 2µ
origin of replication and His3 marker were interchanged for usage in
*Komagataella phaffii*.

Next, we investigated promoters and terminators suitable for expression in
*K. phaffii*. The AOX1 promoter, a methanol inducible promoter, and its
corresponding terminator are used for their high expression rates in
*K. phaffii* [66, 67]. For our project, we decided to build upon the
work of the iGEM Münster team 2022 and use their previously designed vector
pSB3CY\_His ([BBa\_K4188002](http://parts.igem.org/Part%3ABBa_K4188002)) for *S. cerevisiae* (Fig. 33). The pSB3CY\_His has a
*2µ* origin of replication and a *his3* auxotrophy marker. We
replaced both parts with parts commonly used in literature for recombinant
protein expression in *K. phaffii*. Most often, positive transformants
are selected by introducing either an auxotrophy marker or an antibiotic
resistance to the plasmid. The selection of positive transformants in the
project was done by the antibiotic Zeocin®. Many expression vectors
for *K. phaffii* carry *PARS* for replication. Based on the
widespread usage of both Zeocin® for selection and *PARS* as
the origin of replication, these parts were integrated into our vector to
generate the pSB3CYP\_Zeo plasmid ([BBa\_K4675009](http://parts.igem.org/Part%3ABBa_K4675009)) (Fig. 34)­. *Apis mellifera’s vitellogenin* gene was ordered
from IDT in 4 fragments and assembled via Gibson Assembly (Fig. 35).

![The figure shows a Gel electrophoresis of the PCR     amplifying the PARS and zeoR in the constructed pSB3CYP_Zeo. A band at     approximately 700 bp was expected for the amplifications. The analysis     showcased that the parts were integrated into the vector.](https://static.igem.wiki/teams/4675/wiki/results/evvg/3.png)

Figure 34: Gel electrophoresis of the PCR amplifying the PARS and zeoR in
the constructed pSB3CYP\_Zeo (BBa\_K4675009). A band at approximately
700 bp was expected for the amplifications. The analysis showcased
that the parts were integrated into the vector.

![The figure shwos a Gel electrophoresis analyzing     the Gibson Assembly of the Apis mellifera vitellogenin. The gene was assembled     from four fragments synthesized by IDT. A band at 5 kb was expected for the     assembled gene. The correct band was extracted from the gel.](https://static.igem.wiki/teams/4675/wiki/results/evvg/4.png)

Figure 35: Gel electrophoresis analyzing the Gibson Assembly of the
*Apis mellifera* vitellogenin. The gene was assembled from four
fragments synthesized by IDT. A band at 5 kb was expected for the
assembled gene. The band was extracted from the gel.

To begin with the construction of the pSB3CYP\_VgHis10xC, we designed primers
to amplify the *PARS*, *zeoR,* promoter AOX1 and terminator AOX1
from the Open Yeast Library. Additionally, PCRs were carried out amplifying
the *A. mellifera vitellogenin*. The *E. coli* Turbo
strain was used to assemble and amplify our newly constructed plasmids. The
primers were designed to add SapI restriction sites and fusion sites to the
promotor AOX1, the *vitellogenin* gene and the terminator AOX1 for the
assembly into the vector, replacing the *mRFP* gene. Additionally, the
primer designed for *vitellogenin* added a C-terminal 10x His-Tag. The
*zeoR*
was amplified with primers adding NotI and SpeI restriction sites and
*PARS* with primers adding SpeI and BamHI restriction sites. Before
*PARS* could be integrated into the pSB3CY\_His shuttle vector
[(BBa\_K4188002)](http://parts.igem.org/Part%3ABBa_K4188002)
a site-directed mutagenesis had to be performed, deleting a BamHI restriction
site in the PARS origin by changing one base of the sequence. The PCRs were
done using high fidelity polymerase to minimize the introduction of additional
mutations into the DNA sequences. The PCRs amplifying the parts from the
distribution kit did not work and optimization of the primers yielded no
positive results (Fig. 36). Additionally, we tested the plasmids in a gel
electrophoresis for length, to check for dimerization or multimerization. The
gel electrophoresis showcased that nearly all tested plasmid multimerized
during the incubation (Fig. 37). Hence, we decided to order the genes present
in the distribution kit from IDT after discussing the issue with our
instructors and advisors. Using the genes synthesized by IDT as a template,
the fragments containing restriction sites and fusion sites were successfully
amplified. The *zeoR* and *PARS* were integrated into the vector
by restriction cloning, replacing the original *2µ* and *his3*.
The next step was to exchange the *mRFP* with the cassette for
expression of Vitellogenin. The Golden Gate assemblies (RCF[1000]) using the
SapI restriction enzymes did not work using our protocol, as SapI did not seem
to cut in the vector. Here again we saw that dimerization occurred in
*E. coli* Turbo. Shortly here after we switched to
*E. coli* Top10 (Fig. 38). The PCRs for the amplification of the
parts for the expression cassettes were repeated and the protocols checked by
our advisors. Still no successful construction could be achieved. We then
ordered the pSB3CYP\_VgHis10xC vector as fragments and assembled the vector via
Gibson Assembly. We divided the vector into seven fragments and added the
overhangs necessary for the Gibson Assembly directly to the ordered sequences
(Fig. 39). However, sequencing of the construct showcased that the stop codon
in the *zeoR* gene had undergone a mutation, making the gene
non-functional and selection of transformed *K. phaffii* impossible.
Due to time constraints, we were unable to conduct site-directed mutagenesis
in the stop codon of *zeoR*.

![The figure shows a gel electrophoresis of the     PCR amplification for the PARS and ZeoR genes from the plasmids of the     distribution kit. For the amplification of the PARS a band at approximately     200 bp was expected and for zeoR amplification a band at approximately 400 bp.     In the zeoR amplifications one band appeared at 500 bp, which could correspond     to the PCR product. For the PARS amplifications, no bands were detected.](https://static.igem.wiki/teams/4675/wiki/results/evvg/5.png)

Figure 36: Gel electrophoresis of the PCR amplification for the PARS and
ZeoR genes from the plasmids BBF10K 000465 and BBF10K\_000475,
respectively. For the amplification of the PARS a band at approximately
200 bp was expected and for zeoR amplification a band at
approximately 400  bp. In the zeoR amplifications one band appeared
at 500 bp, which could correspond to the PCR product. For the PARS
amplifications, no bands were detected. This gel electrophoresis is
showcased as an overview for all amplifications of the parts used in the
Open Yeast Library.

![The figure shows a Gel electrophoresis checking     the plasmids from the Open Yeast Library. In the first lane is the pAOX1     plasmid, in the second lane is the aMF plasmid, in the third lane is the PARS     plasmid, in the forth lane is the tAOX1 plasmid, in the fifth lane is the zeoR     plasmid, in the sixth lane is the zeoR plasmid. Bands were visible in samples     1;4;5 and 6 at approximately 20 kb. The tested plasmids should be between 2000     bp and 3000 bp in length.](https://static.igem.wiki/teams/4675/wiki/results/evvg/6.png)

Figure 37: Gel electrophoresis checking the plasmids from the Open Yeast
Library. 1= pAOX1 plasmid BBF10K\_000181, 2= aMF plasmid BBF10K\_000355, 3=
PARS plasmid BBF19K\_000465, 4= tAOX1 plasmid BBF10K\_000418, 5= zeoR
plasmid BBF10K\_000475, 6= zeoR plasmid BBF10K\_000475. Bands were visible
in samples 1;4;5 and 6 at approximately 20 kb. The tested plasmids
are between 2000 bp and 3000 bp in length.

![The figure shows a Gel electrophoresis of the       SapI test digestion using the pSB3CY_His vector. The test digestion was done       to check for cutting of the vector by SapI. The first lane is the reaction       mixture containing the plasmid without SapI, the second lane is the reaction       mixture containing the plasmid and SapI,the third lane is the negative control       without plasmid. The pSB3CY_His has a length of 6.3 kb. All samples containing       the plasmids showed a band corresponding to the plasmid, but also a band at 20       kb. No bands were detected corresponding to fragments made during the       restriction digestion. SapI does not seem to cut in the vector.](https://static.igem.wiki/teams/4675/wiki/results/evvg/7.png)

Figure 38: Gel electrophoresis of the SapI test digestion using the
pSB3CY\_His vector ([BBa\_K4188002](http://parts.igem.org/Part%3ABBa_K4188002)). The test digestion was done to check for cutting of the vector by
SapI. 1= reaction mixture containing the plasmid without SapI, 2= reaction
mixture containing the plasmid and SapI, 3= negative control without
plasmid. The pSB3CY\_His ([BBa\_K4188002](http://parts.igem.org/Part%3ABBa_K4188002)) has a length of 6.3 kb. All samples containing the plasmids showed
a band corresponding to the plasmid, but also a band at 20 kb. No
ba  detected corresponding to fragments made during the restriction
digestion. SapI does not seem to cut in the vector.

![The figure shows the       Komagataella phaffii shuttle vector pSB3CYP_VgHis10xC. The plasmid       contains the gene for Vitellogenin and a 10x HisTag at the Vitellogenin       C-terminus.](https://static.igem.wiki/teams/4675/wiki/results/evvg/8.png)

Figure 39: pSB3CYP\_VgHis10xC plasmid map. iGEM part
BBa\_K4675009 was used as the backbone for the construction of the
expression vector. Method for the assembly of the expression vector was Golden
Gate Assembly (RCF[1000]). A C-terminal 10x his-tag was added to the
Vitellogenin for purification of the recombinant protein. For the expression,
*Komagataella phaffii* CBS7450 PEP4 KO was to be used. PARS origin of
replication and Zeocin® resistance (zeoR) were used for selection
of positive *Komagataella phaffii* colonies.

---

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[Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

[Measurement](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D)

[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

{% endblock %}