iGEM 2022 — Team groningen

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{% block title %}Notebook{% endblock %}
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[4 July- 10 July](#1)
[11 July- 17 July](#2)
[18 July- 24 July](#3)
[25 July- 31 July](#4)
[01 August- 07 August](#5)
[08 August- 14 August](#6)
[15 August- 21 August](#7)
[15 August- 21 August](#8)
[22 August- 28 August](#9)
[29 August- 04 September](#10)
[05 September - 11 September](#11)
[12 September - 18 September](#12)
[19 September - 25 September](#13)
[26 September - 2 October](#14)
[3 October - 9 October](#15)
[10 October - 16 October](#16)

# Notebook

*"If you're a bird, I'm a bird"*

## 4 July- 10 July (week 27)

This week we made a mastermix for the replication of the backbone and our G-blocks. All the ordered G-blocks were resuspended. We performed a PCR to amplify the amounts of G-blocks that we had available. Buffers and stocks were made for the agarose gel to check whether the PCR protocol was successful. See the Experiments page for the protocols.

## 11 July- 17 July (week 28)

This week we performed PCR for the amplification of the G-blocks that didn’t work last week. For the successful PCR products we performed a clean-up and checked the yield using Nanodrop (see figure 1). After the clean-up we set up a Golden Gate reaction and made LB plates with ampicillin. Additionally we made competent *E.coli* DH5α cells and the stocks required for the protocol. Finally we transformed the competent cells with the successful Golden Gate constructs, the transformation we checked using a colony PCR. See the Experiments page for the protocols.

| Name | [DNA] | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| R1AB6 nanobody monomer | 40.6 | 1.769 | 2.849 |
| Golden gate AmpR | 6.8 | 1.447 | 1.495 |
| Golden gate anti-GFP nanobody | 43.35 | 1.713 | 3.259 |
| Gibson AmpR | - | - | - |
| Gibson anti-GFP nanobody | - | - | - |
| pYTK001 entry vector | 5.3 | 1.337 | - |

*Figure 1. Nanodrop concentrations for Golden Gate reactions.*

## 18 July- 24 July (week 29)

This week we reperformed last week's colony PCR with a new touchdown protocol (see experiments for protocol). We redid the transformations for the initial experimental anti-GFP & anti-influenza entry plasmids. Then we PCR’ed the GFP3 anti-GFP nanobody and pTRKH3 expression vector, subsequently we performed a clean up and executed a Golden Gate reaction. For the Golden Gate we tested a new protocol (see figure 2), eventually discontinued the protocol in the future. Additionally we also performed a Golden Gate with the anti-influenza nanobody + Ampicillin resistance in the entry vector. From the anti-Influenza ligation we performed a transformation and checked it with a colony PCR and subsequent agarose gel.

| Name | Concentration |
| --- | --- |
| 10x T4 ligase buffer | 1µl |
| T7 ligase | 0,5µl |
| BsAⅠ/BmbⅠ | 0,5µl |
| Backbone (vector) | 20ng |
| R1AB6 nanobody monome | 20ng |
| Golden gate AmpR | 20ng |

*Figure 2. New Golden Gate reaction mixture.*

## 25 July- 31 July (week 30)

This week we tested the purified PCR product from the vector pTRKH3 & R1A-B6-AmpR in pYTK001 (see figure 3) with Nanodrop. We performed a transformation with anti-GFP nanobody in the expression vector pTRKH3. Additionally we started making MRS media for the cultivation of *L.reuteri*.

|  | Name | [DNA] ng/µl | A260/A280 | A260/A230 |
| --- | --- | --- | --- | --- |
| Plasmid | R1A-B6 | 100,9 | 1,865 | 2,247 |
| PCR | R1A-B6 + AmpR | 65,450 | 1,887 | 2,677 |

*Figure 3. Nanodrop concentrations for the anti-influenza nanobodies.*

## 01 August- 07 August (week 31)

This week we tried the initial protein expression experiment with IPTG induction.

## 08 August- 14 August (week 32)

This week we tested the protein mixture of last week's induction by using an SDS-PAGE gel 18% (see Experiments). Also this week we started cultivating *L.reuteri*, made growth curves of *L.reuteri*, and prepared competent cells. We tried the first electroporation (transformation) experiments with *L.reuteri*. Additionally we redid some *E.coli* transformations but this time using the strain BL21.

## 15 August- 21 August (week 33)

This week we tested the transformations of BL21 by means of a colony PCR. We started using vector pUC18 as a control vector to check transformation efficiencies, since our competent cells started to become less competent. Checked competency of both *E.coli* strain (DH5α & BL21). Finally we redid transformation with both *E.coli* strains and *L.reuteri*.

## 22 August- 28 August (week 35)

This week we focussed on the InterLab study. We prepared all the stocks and successfully performed almost all of the transformations in *E.coli* DH5α.

## 29 August- 04 September (week 35)

This week we redid some of the transformations of the Interlab study. Additionally we performed the last plate reading experiments for the InterLab study. For our main project we received newly ordered G-blocks for the kill-switch, which we multiplied. We cleaned up the PCR products, ligated the G-blocks and performed transformations on *E.coli* DH5α & BL21. Started cloning 4 different iterations of the anti-influenza nanobody (2 mono- & 2 bivalent) into the entry and expression vectors. Performed a promoter exchange on the construct pTRKH3 with anti-GFP. Finally we performed a colony PCR on transformed *L.reuteri* strains, from previous weeks, due to slow growth.

## 05 September - 11 September (week 36)

This week we redid *L.reuteri* transformations and checked the kill-switch constructs by means of a colony PCR. We send kill-switch products for sequencing.

## 12 September - 18 September (week 37)

This week we finished the promoter exchange and started with protein expression and purification. We did a transformation with the new promoter exchanged products and send kill-switch parts for sequencing after performing Nanodrop.

## 19 September - 25 September (week 38)

This week we transformed remaining kill-switch components into *E.coli* DH5α & BL21. We made a M9 medium for the kill-switch parts test experiments. Additionally we tested the kill-switch transformation with colony PCR and agarose gel, and sent constructs for sequencing. We performed the initial testing of the kill-switch fluorescence protocol. Finally we started rigorously testing the protein purification protocol.

## 26 September - 2 October (week 39)

This week we performed some of the kill-switch fluorescence experiments, and tested the constructs again using colony PCR. Redid transformations for some of the missing parts of the kill-switch constructs or if they were only available in either *E.coli* DH5α or BL21. Tried to optimize the *L.reuteri* transformation. Tried to optimize the protein expression and purification, performed Western Blot to check if protein was present.

## 3 October - 9 October (week 40)

This week we performed the last plate reader (fluorescence) experiments for the kill-switch. Additionally we performed large scale protein expression and purification for an ELISA experiment.

## 10 October - 16 October (week 41)

This week we colony PCR tested the last *L.reuteri* transformation MRS plates since some growth was detected, samples were put on gel and imaged.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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[Introduction](#introduction)
[Organism Selection](#organism)
[Cloning method: Golden Gate Assembly](#cloning)
[Nanobody expression and secretion](#nanobodyexp)
[Cloning](#cloning1)
[Testing](#testing)
[Biocontainment](#biocontainment)
[Genomic integration](#genomic)
[Engineering cycle](#cycle)
[Part Improvement](#improvement)
[References](#references)

# Engineering

*“The biggest room in the world is the room for improvement.” - Helmut Schmidt*

# Introduction

The goal of our iGEM project at the Rijksuniversiteit Groningen, is to genetically engineer nanobodies against avian influenza that are secreted into lung mucus by native chicken bacteria. To enable this we need to be able to express the nanobodies, secrete them into chicken lungs, and ensure the biosafety by containing its spread during the administration of the therapeutic. The following sections describe engineering details to achieve these goals.

# Organism Selection

To limit microbiome disruption, we require the species of bacteria that expresses and secretes nanobodies to be native to the lungs of chicken. Some other key considerations during selection is its prevalence, survivability, frequency of escape, and potential risks to the lungs of chicken. An added advantage would be if the chosen organism has previously been genetically engineered. A study by Ngunjiri et al. [1], reported the lung microbial species composition of chicken from various age-groups. They did this by extracting the DNA of microorganisms sampled from the trachea, Ileum, nasal and cecum tissues of the lung. This DNA was then sequenced using primers for highly-variable regions of the conserved 16S rRNA, allowing for the identification of present species. Relative abundances of the species could then be determined, resulting in figure 1 below.

*Figure 1. Dynamics of respiratory microbiota across age groups.*

This data (figure 1) showed that while chicken lung microbiomes are highly dynamic, there are some genuses of microorganisms that are quite ubiquitous. For example, various *lactobacillus* species seed all four tissues of the lungs of younger chicken, and remain there quite long as well. In the study this was analyzed in more depth with the same method, resulting in figure 2 below.

*Figure 2. Ecology of Lactobacillus in URT and LT sites. Bar charts show changes in relative abundance of genus-level bacterial taxa (vertical axis) with chicken layer age (horizontal axis). 01W, 03W etc. indicate ages of birds in weeks. Nasal, nasal cavity.*

As can be seen, various species such as *L. crispatus, L. gasseri, L. reuteri, L. salivarius,* and *L. vaginalis* are found long-term in all 4 analyzed regions of the lungs, and are therefore good candidates for nanobody expression.

Beyond the ability to colonize the lungs, the ease of modification (including precedence, so whether there are existing protocols), effectiveness of (nanobody) protein expression, and its effects on chicken health are also important for our purposes. Further literary research showed that *L. reuteri* specifically was an ideal candidate, as it already fulfills a beneficial role in lung microbiome health through its production of reuterin, a antimicrobial molecule which protects lung mucus from foreign bacteria. Moreover, the organism has been previously used to express and secrete nanobodies already, like in a study by Krüger et al. [2], where nanobodies against *Streptococcus* mutans were expressed and secreted using *L. reuteri* for the purpose of in-situ passive immunization against the pathogen . This was in essence the same principle as we intended to do with avian influenza, hence we adopted some of the methods used in these studies to allow for streamlined modification of *L. reuteri*.

# Cloning method: Golden Gate Assembly

The construction of assemblies described below was performed through Golden Gate Assembly. To use this method on a region of DNA a set of primers designed containing 3’ annealing regions complementary to the beginning and end of the target region (as with any PCR), but also contain a 5’ tail region containing a 3’ pointing type IIS restriction site. When these primers are used, the tails create 5’ and 3’ inward-pointing restriction sites which, when cut with the restriction enzyme, cleave off the restriction recognition sites. When an insert region and a vector are PCRed using this method and combined in a one-pot mixture with the restriction enzyme and ligase, the two fragments can be assembled in a scarless manner. In a PCR machine the restriction enzyme will first cut off the restriction recognition sites of both fragments, producing sticky-ends compatible with one-another as well as with the small cut off recognition site. The temperature is changed allowing for ligase to function and ligate the insert and vector fragment, or the fragments with the cut-off recognition sites. If the latter occurs it will be possible for the restriction enzyme to function again in the next round, though if the former occurs the restriction recognition site is no longer present and the final construct will have been assembled. Through many rounds this will result in the specific assembly of the added fragments in one pot.

This pot can then be immediately transformed and plated on selection plates. This process is illustrated below (figure 3).

*Figure 3. Specific assembly of the added fragments in one pot.*

Within this project, this method has been adapted to assemble all constructs, using the type IIS restriction enzyme BsaI, capable of cutting as follows: GGTCTC(1/5), thus generating a 4 bp overhang. Though a number of Golden Gate components were synthesized de novo, through the IDT DNA synthesis service [4], a number were PCRed using primers designed to insert BsaI restriction recognition sequences pointing inwards at the ends of the DNA fragments. The overhangs formed by these restriction enzymes vary per component, but have been standardized to allow for modularity amongst many of the constructs, as will be described in more detail in later sections.

# Nanobody expression and secretion

In a stepwise fashion, first a fitting organism must be selected, followed by an investigation of how this organism could secrete small proteins like nanobodies. Then this protein secretion mechanism must be adapted to the nanobodies in (preferably) one plasmid, which can then be transformed into the selected organism.

In practice, this would result in the general assembly of: Vector, promoter, nanobody with fused tags, terminator. Though the organization of this depends heavily on precedence, allowing for more effective comparative analyses.

Once the construct is built an investigation into the expression and secretion efficiency of the nanobody must then follow, as well as the elucidation of the effectiveness of nanobody binding to the desired target. Both are also to be compared to an easily analyzed control nanobody.
With the data acquired from the respective experiments it will be possible to determine the efficacy and feasibility of the nanobody-based therapeutic probiotic strain we hope to create.

## Design considerations

### Vector selection

Once a fitting host organism was selected it was a matter of finding past research groups which had worked with *L. reuteri* to express and secrete nanobodies. This was found in a study by Lizier et al. [5], where a comparative analysis between different expression vectors was performed on various *L. reuteri* strains. This was done by transforming pTRKH3-based vectors powering EGFP with 3 different promoters (ErmB, ldhL, slp) into *Lactococcus lactis spp. Cremoris* MG1363, *Limosilactobacillus reuteri* DSM 20016, and 5 different *L. reuteri* strains from chicken crops. The results showed that significant expression of EGFP can be achieved by the pTRKH3 vector in various strains, and especially in *L. reuteri* DSM20016, where regardless of the used promoter it resulted in good protein expression.

*Figure 4. Vector selection.*

With further research it was found that the pTRKH3 vector contains both an origin of replication (Ori) that is functional in *E. coli*, as well as one that allows for replication in *L. reuteri*. Though the latter at lower copy numbers, this will limit the metabolic load nanobody production would have on the organism, likely improving survivability in lung mucous. The identification of the nature of the ori in this plasmid was difficult, but through the use of nBLAST we determined it to be based on the pAMβ1 replication mechanism [6].

Additionally, since the vector contains a strongly expressed GFP gene, replacing this with whatever insert is desired will allow for easy recognition of false positives, since successful replacement changes colony color from fluorescent green to colorless. Therefore, this vector appears ideal for expression of the nanobody, and was chosen as the basis of our engineering.

Though this expression vector will be used for the final construct in *L. reuteri*, the pYTK001- and pYTK095 vectors from the Yeast Toolkit for Modular Assembly by Lee et al. [7], will be used as control and cloning backbones . These plasmids were designed to carry standardized Golden Gate Assembly constructs and contain a chloramphenicol and ampicillin resistance gene respectively, a p15 ori, and a superfolder GFP gene. This plasmid is far smaller (2676 bp) that the pTRKH3 expression vector (8961 bp) and has a very high copy number, thus allowing for more efficient cloning in *E. coli* and expression, on top of easy recognition of false positives when GFP is expressed.

## Nanobody expression and secretion cassette

For the nanobody cassette itself several components are of importance. First a promoter, then the nanobody itself with fused tags, and finally a terminator. With this basic set-up both nanobody expression plasmids are designed.

First for the promoter, Lizier et al. [5], already compared three promising candidates, though these may be too strong for our purposes. It was therefore decided to use the weak constitutive Lacl promoter, as well as the IPTG inducible T7 promoter, the former being intended for final product use, and the latter being useful for expression and secretion testing.

### Nanobody selection

For the final product the organism must express and secrete anti-hemagglutinin nanobodies. A number of candidates for our project revealed themselves in a study by Hufton et al. [20], where they generated and characterized 18 single-domain VHH nanobodies against the 2009 A(H1N1) avian influenza virus hemagglutinin through the use of an immune alpaca phage display library. A number of these nanobodies were binding but non-neutralizing, while others only neutralized H1N1, but most interestingly were the 5 variants with broad cross-subtype neutralizing capacity. Amongst these, variants R1a-B6 and R1a-A5 had consistently strong binding and neutralization in various avian influenza subtypes (see figure 5), and were therefore chosen as proof-of-concept nanobodies in our nanobody expression and secretion platform.

*Figure 5. Binding of nanobody variants.*

Additionally, the study experimented with turning R1a-B6 and R1a-A5 bivalent, which both increased neutralization efficacy to avian influenza variant A(H9N2) and gained the ability to neutralize variant A(H2N2). Therefore we decided to also test both of these nanobodies in parallel, using the (G5S)6 linker fused in between the two nanobodies and generating the variants R1a-B6-linker-B6, R1a-A5-linker-A4, and R1a-B6-linker-A5.

To confirm the ability of *L. reuteri* to produce functional nanobodies with our construct we also designed a control which produces anti-GFP nanobodies. This protein, which was originally developed by Rothbauer et al. [8], was selected based on a study by Kubala et al. [9], where a structural and thermodynamic analysis of the GFP:anti-GFP-nanobody complex was performed through x-ray crystallography and isothermal titration calorimetry respectively, showing tight binding of the nanobody to the fluorescent protein. With such well known binding, this anti-GFP nanobody is a perfect candidate as a control of nanobody expression and binding.

*Figure 6. Interaction analysis of the GFP;GFP-nanobody complex.*

This protein was expressed using the *E. coli* strain BL21-GOLD by Kubala et al. [9], we therefore codon optimized the gene for *L. reuteri* DSM20016. This was done using the OPTIMIZER tool created by Puigbò et al.[10], using the *L. reuteri* codon usage tablefrom the Kazusa codon usage database [11]. An added advantage of this is that we could express with less efficiency the codon optimised reuteri genes in E.coli.

### Nanobody expression cassette design

Beyond the nanobody coding regions themselves, several additional aspects were considered during the design of the nanobody expression cassette, including the promoter, terminator and fused tags.

First, the promoter needed to be inducible to most effectively show whether nanobody expression has occurred. As one of the most commonly used promoters, the IPTG-inducible lac promoter was chosen for this purpose, for its well characterized nature, its relatively strong activity, as well as its common, effective and cheap induction chemical [12].

Next, besides nanobody expression, one of the goals of our project is the secretion of these peptides into extracellular space. For this purpose *L. reuteri* recognizes secretion signals which help guide proteins out of the cell. Therefore we chose the Cnb (Collagen-binding protein) secretion signal peptide for fusion to the N-terminal end of the nanobody, based on the comparative analysis of various *L. reuteri* secretion signals by Liu et al. [13]. In this study the efficacy of 29 secretion signals was analyzed by fusing them to lactonohydrolase from *Rhinocladiella mackenziei*, which is capable of hydrolyzing the mycotoxin ZEN (zearalenone). In this study, the Cnb secretion signal, here named S9, was found to be the most effective.

*Figure 7. The signal peptide screening for the secretion expression system in L. reuteri.*

Besides the secretion signal, it was also decided to fuse a 6x His-tag to the C-terminal end of the nanobodies to allow for more effective protein purification, which will become vital during the expression and secretion analysis. And for the anti-hemagglutinin constructs a Myc tag is also fused to the C-terminal end of the nanobody, which allows for an ELISA to be performed to confirm nanobody binding efficiency of our final products. Antibodies against this tag have already been developed long ago, for example by Evan et al. [14].

Lastly a terminator is required to end transcription. Though a number of efficient terminators are available, due to its ubiquitous and reliable nature it was decided to use the double terminator from biobrick BBa\_B0015. This terminator efficiently ends transcription from both directions, which will become important in other constructs.

The end product of these assemblies are shown below in figure 8, where the arrangement of the expression cassettes are shown.

*Figure 8. Arrangement of the expression cassettes.*

# Cloning

As mentioned above, various components are to be combined to produce the final nanobody expression and secretion plasmid. These components have been generated in 2 ways, PCR of existing plasmids or biobricks, and de novo synthesis. In this chapter the generation of these components will be described, as well as the assembly thereof into composite parts and plasmids.

## Entry vector backbone

The entry vector used for the nanobody expression and secretion section of Nanobuddy was pYTK001, where PCR with p14 and p15 excised the superfolded GFP, amplifying the pMB1 ori and CmR, generating BBa\_K4233000 as seen below in figure 9.

*Figure 9. Creation of the Entry vector backbone for nanobody production and secretion BBa\_K4233000.*

## Expression vector backbone

The expression vector used here was pTRKH3, where PCR with p5 and p6 excised mGFP5 and TetR, amplifying EryR, the pAMβ1 ori, and the p15 ori, generating BBa\_K4233001 as seen below in figure 10.

*Figure 10. Creation of the expression vector for nanobody production and secretion BBa\_K4233001.*

## Antibiotic resistance marker

For selection of successful assemblies, an AmpR gene was also used. This fragment was synthesized de novo based on the AmpR from pYTK095. No PCR was used here, as in this stage of experimentation we did not have access to the pYTK095 plasmid, only the sequence. This synthesis generated BBa\_K4233002, as seen below in figure 11.

*Figure 11. Creation of the antibiotic resistance marker AmpR BBa\_K4233002.*

## Anti-GFP nanobody expression cassette in the entry vector

For the assembly of the initial anti-GFP nanobody expression cassette entry vector construct, the BBa\_K4233000 entry vector fragment, de novo synthesized BBa\_K4233003 anti-GFP nanobody cassette, and BBa\_K4233002 ampicillin resistance marker were Golden Gate Assembled as outlined before. This generated pRUC2001 (BBa\_K4233008), as seen below in figure 12.

*Figure 12. Assembly of anti-GFP nanobody expression cassette in the entry vector BBa\_K4233008.*

## Anti-GFP nanobody expression cassette in the expression vector

For the assembly of the initial anti-GFP nanobody expression cassette expression construct, the BBa\_K4233001 expression vector fragment, de novo synthesized BBa\_K4233003 anti-GFP nanobody cassette, and BBa\_K4233002 ampicillin resistance marker were Golden Gate Assembled as outlined before. This generated pRUC2006 (BBa\_K4233013).

In addition, after expression-levels of the nanobodies with the lac promoter was deemed suboptimal, promoter exchange was attempted on BBa\_K4233013 by performing PCR with p5 and p65, resulting in intermediate part BBa\_K4233082, then circularizing this fragment with Golden Gate Assembly. This would have generated pRUC2206-T7 (BBa\_K4233014), but cloning of this construct could not be performed in time (figure 13).

*Figure 13. Assembly of anti-GFP nanobody expression cassette in the expression vector BBa\_K4233014.*

## Anti-hemagglutinin nanobody expression cassette in the entry vector

For the assembly of the initial anti-hemagglutinin nanobody expression cassette entry vector constructs, the BBa\_K4233000 entry vector fragment, de novo synthesized BBa\_K4233004/BBa\_K4233005/BBa\_K4233006/BBa\_K4233007/BBa\_K42330077 anti-hemagglutinin nanobody cassettes, and BBa\_K4233002 ampicillin resistance marker were Golden Gate Assembled as outlined before. This generated the pRUC2002 (BBa\_K4233009), pRUC2003 (BBa\_K4233010), pRUC2004 (BBa\_K4233011), pRUC2005 (BBa\_K4233012) and pRUC2036 (BBa\_K42330078) plasmids. Of these constructs the BBa\_K4233077 and BBa\_K4233078 constructs were conceptual due to synthesis issues (figure 14).

*Figure 14. Assembly of anti-hemagglutinin nanobody expression cassette in the entry vector BBa\_K4233009 till BBa\_K4233012 and BBa\_K4233078.*

## Anti-hemagglutinin nanobody expression cassette in the expression vector

For the assembly of the initial anti-hemagglutinin nanobody expression cassette expression vector constructs, the BBa\_K4233001 expression vector fragment, de novo synthesized BBa\_K4233004/BBa\_K4233005/BBa\_K4233006/BBa\_K4233007/BBa\_K42330077 anti-hemagglutinin nanobody cassettes, and BBa\_K4233002 ampicillin resistance marker were Golden Gate Assembled as outlined before. This generated the pRUC2007 (BBa\_K4233015), pRUC2008 (BBa\_K4233016), pRUC2009 (BBa\_K4233017), pRUC2010 (BBa\_K4233018) and pRUC2037 (BBa\_K4233079) plasmids. Again, since BBa\_K4233077 could not be synthesized, BBa\_K4233079 could not be constructed.

Here too T7 promoter exchange was performed on all constructs using PCR with p5 and p65, generating the linear fragments BBa\_K4233083, BBa\_K4233084, BBa\_K4233085, BBa\_K4233086 and BBa\_K4233087 (conceptual). Though the Golden Gate Assembly of these fragments into the pRUC2007-T7 (BBa\_K4233019), pRUC2008-T7 (BBa\_K4233020), pRUC2009-T7 (BBa\_K4233021) and pRUC2010-T7 (BBa\_K4233022) plasmids was attempted, a lack of time prevented its completion. Plasmid pRUC2037-T7 (BBa\_K42330080) is again, conceptual due to the lack of BBa\_K4233079 (figure 15).

*Figure 15. Assembly of anti-hemagglutinin nanobody expression cassette in the expression vector BBa\_K4233015 till BBa\_K4233022, BBa\_K4233079, and BBa\_K4233080.*

# Testing

## Assembly confirmation

Once these constructs had been assembled they were transformed into *E. coli* DH5ɑ and plated on LB plates with the relevant antibiotics. The resulting colonies were tested with colony PCR and brief exposure with UV light to determine assembly success (as false positives result in green fluorescence from the superfolded GFP of the uncut vectors), and those that pass this round were be inoculated in LB medium and the plasmids harvested for sequencing. When a successfully assembled plasmid had been found it was transformed, through electroporation, into *L. lactis* DSM20016 and *E. coli BL21*, after which expression and secretion was tested.

## Nanobody secretion testing

Secretion testing will occur in L.reuteri since by design the secretion signal is codon optimized for our specific strain (L.reuteri DSM20016). However creation of the specific constructs will be performed in E.coli DH5α and E.coli BL21, since E.coli is much easier to cultivate than L.reuteri. According to literature L.reuteri should be a facultative anaerobic bacteria, but for easy and fast growth we chose for the molecular workhorse E.coli.
The secretion testing itself is based on two primary research questions. Can the expression vector effectively use the nanobody cassette to express the nanobody in vivo? And does the secretion signal work effectively in the host bacteria?

To elucidate this, the L. lactis DSM20016 containing the expression and entry vectors with the nanobody cassettes will be used for protein expression, and the resulting cultures are then pelleted through centrifugation. The supernatant and pellet will be collected separately and the latter is disrupted through sonification to maintain protein structure integrity. The protein suspensions are centrifuged again to remove any remaining cell debris and used for SDS-PAGE and western blot to determine the presence of the nanobodies inside and outside the cells. This will both show in principle the viability of expression of the nanobodies and the subsequent secretion thereof.

For testing the strength of the secretion signal we would collect again protein suspensions from the supernatant and the cell lysis mixture. However instead of performing SDS-PAGE or western blot, we would firstly perform protein purification with a Nickel beads column and a Bradford assay with a BSA control. From this we should be able to determine purified protein concentrations from both inside and outside the cells, and thus do a fractional analysis to conclude on the strength of the secretion signal.

## Nanobody efficacy testing

When expression and secretion is determined to be successful, the nanobody binding efficiency can be quantified using direct ELISA (see figure 16 below) [15, 16]. The nanobodies contain MIC tags and his-tags, which both have known antibodies with bound reporter enzymes in this case Horseradish peroxide (HRP). By coating the 96 wells maxisorp plates with GFP and hemagglutinin proteins and incubating them with known concentrations of the anti-GFP and anti-hemagglutinin nanobodies, the binding is facilitated. Then by subsequent incubation of the secondary antibodies which are already bound to the reporter enzymes and the addition of a chemiluminescent substrate after washing. We are able to determine the binding affinity of previously mentioned antibodies respectively based on the fluorescence of the converted chemiluminescent substrate.

*Figure 16. Direct ELISA to quantify the nanobody binding efficiency*

# Biocontainment

Though the primary goal of this project was to genetically modify *L. reuteri* to express and secrete anti-influenza nanobodies, its in situ application will both involve spraying of the organism into the environment, and its subsequent survival exclusively in the lungs of chickens. Since it is possible for bacteria to incorporate foreign DNA into its own genome, it would be possible for the DNA of a genetically engineered organism to enter the environment and be absorbed and utilized by other species in unexpected ways. This is unlikely to have harmful effects, but since this is not an absolute certainty, it is wise to limit the release of modified organisms and their DNA into nature, which is no different with this project.

## Design considerations

So to ensure that the organism only survives inside chicken lungs a killswitch mechanism was designed using two environmental factors, high temperature and the absence of light. After all, chicken lungs are both warm (41℃) and dark, which are unusual environmental factors not often found outside living organisms, especially in the Netherlands. So when these mechanisms are employed, only those two factors will allow the organism to not die.

Usually utilizing only one of these two is enough, however due to the non-binary activation behaviour of temperature and light switches, and the decreased likelyhood of escape when using to separate killswitches, both were designed in parallel but can also be used individually.

### Killing mechanism

As mentioned above, the primary goal of the kill-switches is to kill the microorganism under specific circumstances. Naturally this requires a killing mechanism, of which a number of possible variants are available. One more well known type is a toxin/antitoxin mechanism, where a toxin is constitutively expressed while an antitoxin is activated by some external signal. Though this is a well documented method, it is not always the most effective. Therefore we decided it was interesting to try a CRISPR-Cas9-based killing mechanism, which could have the capacity for greater killing efficiency and greater adaptability.

This was explored in a study by Rottinghaus et al [17], where a constitutive Pcon promoter controls a tetR gene, which inhibits constitutive Ptet promoters controlling a gRNS and a Cas9 gene. TetR is inhibited by aTc (anhydrotetracycline), so adding this molecule to the organism containing the construct would result in the inability of TetR to inhibit the Ptet promoters, subsequently allowing gRNA and Cas9 to express and to form a CRISPR-Cas9 complex, killing the cell (figure 17).

![](https://static.igem.wiki/teams/4233/wiki/engineering-17.png)

*Figure 17. Schematic of the CRISPR-Cas9 kill mechanism by Rottinghause et al [17].*

Kill mechanism testing showed a significantly decreased colony forming unit count when aTc was added, though the effectiveness varied based on the specific target selected. Therefore it was chosen to have our own sgRNA target various conserved regions of *L. reuteri* DSM 20016 16S rRNA, and compare effectiveness, as this gene is highly conserved in bacterial species to begin with, allowing for the potential adaptation of the kill switches into species other than *L. reuteri*.

Similar to our intent to seed the organism in chicken lungs, the paper also tested in vivo killswitch effectiveness, by seeding the gut of mice with their modified *E. coli* Nissle 1917 strain. Feeding the mice with aTc resulted in the rapid decrease in modified *E. coli* CFUs recovered from feces, as can be seen in Figure 18 below.

*Figure 18. Effectiveness of the kill mechanism in vivo.*

### Light killswitch

In order to utilize our chosen kill mechanism we need a way to convert environmental stimuli into activation of expression of the cas9, while the sgRNA is constantly transcribed. As said before one of the chosen environmental factors is light, however because of the nature of photons, light is difficult to detect and often requires cofactors not usually synthesized by bacteria. To engineer a bacterium to allow for the synthesis of these cofactors frequently requires many different enzymes and thus cloning steps which would not be achievable within this project. This is why we use the natural ability of *L. reuteri* to produce cobalamin (Vitamin B12), which can detect light and together with the transcription factor CarH has the ability to regulate gene expression. CarH is a bacterial transcription factor used by Myxococcus xanthus and Thermus thermophilus, which binds Adenosylcobalamin (a form of B12 produced by *L. reuteri*) and forms a tetramer allowing the complex to bind DNA and thus stop transcription (figure 19). Light triggers the dissociation of the tetramer and thus allows gene expression.

![](https://static.igem.wiki/teams/4233/wiki/engineering-19.png)

*Figure 19. Schematic visualization of the dissociation of the CarH tetramer triggered by light.*

In order to combine the light sensor and kill mechanism into a light activated killswitch we designed the cassette as in figure 20. Here we placed the constitutive strong promoter J23102 to produce the TtCarH repressor derived from Thermus thermophilus, which in turn represses pCarH promoter activity localized upstream of Cas9. When exposed to light the CarH will dissociate and release the promoter allowing transcription of the Cas9 which will bind the sgRNA constitutively transcribed by J23100 and start cutting the 16S rRNA which will kill the cell. J23100 is used to control constitutive sgRNA transcription, because in nature it controls shRNA (small hairpin RNA) transcription, and therefore does not add the poly-A tail to the RNA which would normally mediate degradation of the molecule. This also positions the sgRNA at the end of the cassette allowing for easy substitution when this cassette is utilized in a different organism.

*Figure 20. scheme of the light killswitch cassette BBa\_K4233069.*

### Temperature killswitch

The other variable we looked at to distinguish between the environment of the chicken lung and the outside world is temperature. The body temperature of a chicken is between 40 and 42 degrees celsius whereas the outside temperature, especially in the Netherlands, will rarely reach this high. We decided upon the TlpA system derived from Salmonella typhimurium. This protein forms a dimer which binds its associated promoter and stops transcription, however at high temperatures the dimer becomes unstable releasing the promoter and allowing gene expression. The main upside of this system is that the transition temperature is around 41 degrees celsius which is similar to the body temperature of the chicken but not a human, and results in a 200-fold increase in expression. Furthermore this system has previously been used to create a kill switch in *E. coli* and has already been engineered to transition at 39 and 36 degrees celsius by Piraner et al [18], which could prove useful in fine tuning the system. To further tune the system we test 2 different promoters, one is the native promoter of TlpA and the other an adapted version. This is because the native promoter includes a peculiar region between the -35 and -10 boxes consisting of only 2 nucleotides per strand, guanine and thymine on the one side and cytosine and adenine on the other. The reason for this is unclear but it is hypothesized that it aids the binding of TlpA dimer. The adapted version of the promoter changes this region for the same region of the pbu promoter, a promoter native to *L. reuteri* and involved in the genes responsible for glycerol metabolism. We hypothesize that this change will decrease binding of the protein and thus cause more leakage but also provide a higher uninhibited expression of the gene, this will then serve as a proof-of-concept that these parameters can be fine tuned to create a more effective and safe use of our treatment.

*Figure 21. Schematic visualization of the regulation of pTlpA at increasing temperature.*

As can be seen in the figure 21 above this system is deactivated at low temperature and activates as the temperature increases. If we were to use the same cassette setup as with the CarH system we would get the opposite result to what we want to achieve, the bacteria will survive at low temperatures while it dies as it enters the lung. This is why we need to invert the signal, which can be done using TetR. TetR is a protein that represses the ptet promoter, and thus can invert a signal resulting in the activation of the kill mechanism at low temperatures.

We designed the temperature killswitch cassette based on the design by Piraner et al [18], as this has been proven to work. Accordingly the 3 TlpA variants of TlpA36, TlpA39, and TlpA41 are coexpressed with TetR by the 2 variants of the pTlpA promoter mentioned before. This creates 6 variants of the killswitch showing the adaptability of the system. The TetR is expressed at high temperatures and proceeds to inhibit the Tet promoter, but at low temperatures the TetR is repressed allowing for expression of the Cas9. The Cas9 then binds the constitutively expressed sgRNA and proceeds to kill the cell (figure 22).

*Figure 22. Scheme of the temperature killswitch cassettes BBa\_K4233063 till BBa\_K4233068.*

## Cloning strategy

As mentioned above, various components are to be combined to produce the final killswitch plasmids. These components have been generated in 2 ways, PCR of existing plasmids or biobricks, and de novo synthesis. In this chapter the generation of these components will be described, as well as the assembly thereof into composite parts and plasmids

### Entry vector backbone

The Entry Vector used in creating the kill switch for Nanobuddy was pYTK095. The superfolded GFP has been excised by PCR using p48 and p50 to generate BBa\_K4233025, p49 and p50 to generate BBa\_K4233026, or p50 and p62 to generate BBa\_K4233027. All backbones include the AmpR and pMB1 ori, while only the later two also include the BBa\_B1005 terminator. The difference between BBa\_K4233026 and BBa\_K4233027 is the BsaI cut-site used during Golden Gate Assembly, as can be seen below in figure 23.

*Figure 23. Creation of kill switch Entry vector backbone BBa\_K4233025 till BBa\_K42330.*

### Expression vector backbone

The expression vector was created from pTRKH3-ermGFP, using p5 and p65 to excise the GFP, TetR and EryR and amplify the pAMβ1 ori and p15 ori. This generates part BBa\_K4233029, as can be seen below in figure 24.

*Figure 24. Creation of kill switch expression vector backbone BBa\_K4233029.*

### Antibiotic resistance marker

The antibiotic resistance marker used to create the killswitch is PCRed from pYTK001 using p51 and p52, this generates part BBa\_K4233028 as can be seen below in figure 25.

*Figure 25. Creation of antibiotic resistance marker fragment creation BBa\_K4233028.*

### mRPF1

The mRFP1 fragments used to test the promoters of the killswitch were created using two different methods. The biobrick BBa\_K516030 was PCRed using p30 and p31 to create a fragment containing the mRFP1 and a double terminator (BBa\_K4233023). While the other fragment containing the mRFP1 and the BBa\_B1002 terminator was synthesized de novo generating part BBa\_K4233024 (figure 26).

*Figure 26. Creation of mRFP1 fragments BBa\_K4233023 and BBa\_K4233024.*

## Test Plasmids

We now know out of which promoters our killswitch will be built, but before this system can be assembled its promoters first need to be tested. We did this by making them express mRFP1 and measuring the fluorescence. This gives us insight into the strength and transition values of the promoters.

### Constitutive promoter test

For the assembly of both constitutive promoter test plasmids the promoters BBa\_J23100 and BBa\_J23102 were de novo synthesized to generate parts BBa\_K4233030 and BBa\_K4233031 respectively. These were combined with the BBa\_K4233023 mRFP1 fragment and the BBa\_K4233025 backbone to create pRUC2211 (BBa\_K4233041) and pRUC2212 (BBa\_K4233042), as shown below in figure 27.

*Figure 27. Assembly of the constitutive promoter test plasmids BBa\_K4233041 and BBa\_K4233042.*

### TetR test

For the assembly of the TetR test plasmid a fragment containing the J23102 promoter, the TetR gene, a double terminator, and the Tet promoter was synthesized to generate part BBa\_K4233033. This was combined with the BBa\_K4233024 mRFP1 fragment and the BBa\_K4233026 backbone to create pRUC2214 (BBa\_K4233044), as shown below in figure 28.

*Figure 28. Assembly of the TetR test plasmid BBa\_K4233044.*

### TlpA test

For the assembly of the TlpA test plasmids the fragments containing all 6 combinations of the 2 promoters and the 3 TlpA variants were synthesized to generate parts BBa\_K4233034, BBa\_K4233035, BBa\_K4233036, BBa\_K4233037, BBa\_K4233038, and BBa\_K4233039. These were combined with the BBa\_K4233023 mRFP1 fragment and the BBa\_K4233025 backbone to create pRUC2215 (BBa\_K4233045), pRUC2216 (BBa\_K4233046), pRUC2217 (BBa\_K4233047), pRUC2218 (BBa\_K4233048), pRUC2219 (BBa\_K4233049), and pRUC22120 (BBa\_K4233050) respectively, as shown below in figure 29.

*Figure 29. Assembly of the TlpA test plasmids BBa\_K4233045 till BBa\_K4233050.*

### CarH promoter test

For the assembly of the CarH promoter test plasmid a fragment containing the pCarH promoter should have been synthesized generating part BBa\_K4233032. However it could not be synthesized and could not be optimized due to time constraints, but for further inquiries we recommend contacting the writers of this page. If obtained, the part would have been combined with the BBa\_K4233023 mRFP1 fragment and the BBa\_K4233025 backbone to create pRUC2213 (BBa\_K4233043) , as shown below in figure 30.

*Figure 30. Assembly of the CarH promoter test plasmid BBa\_K4233043.*

### CarH test

For the assembly of the CarH test plasmid a fragment containing the J23102 promoter, the CarH gene, and the pCarH promoter should have been synthesized generating part BBa\_K4233040. However as said before this was not possible within the given time frame. If obtained, the part would have been combined with the BBa\_K4233024 mRFP1 fragment and the BBa\_K4233026 backbone to create pRUC2221 (BBa\_K4233051), as shown below in figure 31.

*Figure 31. Assembly of the CarH test plasmid BBa\_K4233051.*

## Final temperature kill-switch

The final kill-switches are assembled in 3 steps: The kill-switch cassette assembly, the kill-switch cassette amplification, and then kill-switch expression vector incorporation.

### Temperature kill-switch cassette assembly

To assemble the final temperature killswitch cassette, several components are needed. First are the 5 temperature sensor fragments, pTlpA1-TlpA36C (BBa\_K4233034), pTlpA1-TlpA39C (BBa\_K4233035), pTlpA1-TlpA41C (BBa\_K4233036), pTlpA2-TlpA36C (BBa\_K4233037), pTlpA2-TlpA39C (BBa\_K4233038) and pTlpA2-TlpA41C (BBa\_K4233034). Next, TetR inverter component BBa\_K4233033 is PCRed using p34 and p35 to remove the constitutive J23102 promoter. Biobrick BBa\_2457006 (from iGEM17\_Amazonas\_Brazil) is PCRed using p55 and p57 to amplify only the Cas9 coding region, and to add a downstream BBa\_B1002 terminator, generating BBa\_K4233053. Lastly the sgRNA cassette targeting *L. reuteri* 16S rRNA has been synthesized de novo. All these components are used in a Golden Gate Assembly with entry vector fragment BBa\_4233027 to form pRUC2222 (BBa\_K4233056), pRUC2223 (BBa\_K4233057), pRUC2224 (BBa\_K4233058), pRUC2225 (BBa\_K4233059), pRUC2226 (BBa\_K4233060) and pRUC2227 (BBa\_K4233061) respectively. See figure 32 below.

*Figure 32. Assembly of temperature kill-switch cassette BBa\_K4233056 till BBa\_K4233061.*

### Temperature kill-switch cassette amplification

Once all the individual components have been assembled into pRUC2222-pRUC2227, these plasmids can be used as a template to amplify the entire killswitch cassette. This region, from the temperature sensor component to the sgRNA cassette are to be PCRed using p36 and p59, generating the killswitch cassette fragments pTlpA1-TlpA36C (BBa\_K4233063), pTlpA1-TlpA39C (BBa\_K4233064), pTlpA1-TlpA41C (BBa\_K4233065), pTlpA2-TlpA36C (BBa\_K4233066), pTlpA2-TlpA39C (BBa\_K4233067) and pTlpA2-TlpA41C (BBa\_K4233068). See figure 33 below.

*Figure 33. Amplification of Temperature kill-switch cassette BBa\_K4233063 till BBa\_K4233068.*

### Temperature kill-switch expression vector incorporation

Finally, with the temperature killswitch cassettes amplified, they can be incorporated into the expression vector. This is done by performing Golden Gate Assembly on the cassettes pTlpA1-TlpA36C (BBa\_K4233063)/pTlpA1-TlpA39C (BBa\_K4233064)/pTlpA1-TlpA41C (BBa\_K4233065)/pTlpA2-TlpA36C (BBa\_K4233066)/pTlpA2-TlpA39C (BBa\_K4233067)/pTlpA2-TlpA41C (BBa\_K4233068), the chloramphenicol resistance marker BBa\_K4233028, and the expression vector BBa\_K4233029. The resulting constructs are pRUC2229 (BBa\_K4233070), pRUC2230 (BBa\_K4233071), pRUC2231 (BBa\_K4233072), pRUC2232 (BBa\_K4233073), pRUC2233 (BBa\_K4233074) and pRUC2234 (BBa\_K4233075) respectively, as can be seen below in figure 34. These are the final kills-switch constructs ready for testing.

*Figure 34. Assembly of temperature kill-switch expression vector BBa\_K4233070 till BBa\_K4233075.*

## Final light kill-switch

Here too, the final kill-switches are assembled in 3 steps: The kill-switch cassette assembly, the kill-switch cassette amplification, and then kill-switch expression vector incorporation. The assembly of the light kill-switch is highly similar to the temperature kill-switches, and differs only in the sensor component and the overhangs used for the Cas9 component. It will therefore be described in a more brief fashion.

### Light kill-switch cassette assembly

The light kill-switch cassette was assembled using the de novo synthesized CarH light sensor component (BBa\_K4233040), the Cas9 coding region amplified using p56 and p57, the sgRNA cassette (BBa\_K4233055) and the entry vector (BBa\_K4233027), to form the plasmid pRUC2228 (BBa\_K4233062) as shown below in figure 35.

*Figure 35. Assembly of light kill-switch cassette BBa\_K4233062.*

### Light kill-switch cassette amplification

The light kill-switch cassette is then amplified by performing PCR on pRUC2228 using p34 and p59, generating fragment BBa\_K4233069 as shown below in figure 36.

*Figure 36. Amplification of light kill-switch cassette BBa\_K4233069.*

### Light kill-switch expression vector incorporation

Finally, with the light killswitch cassette amplified, it can be assembled into the final light kill-switch through Golden Gate Assembly with the chloramphenicol resistance marker BBa\_K4233028 and the expression vector BBa\_K4233029, to form pRUC2225 (BBa\_K4233076) as can be seen below in figure 37. This is the final light kill-switch construct ready for testing.

*Figure 37. Assembly of light kill-switch expression vector BBa\_K4233076.*

## Testing

### Assembly confirmation

Once these constructs had been assembled they were transformed into *E. coli* DH5ɑ and plated on LB plates with the relevant antibiotics. The resulting colonies were tested with colony PCR and brief exposure with UV light to determine assembly success, as false positives result in green fluorescence from the superfolded GFP of the uncut vectors, while successful assemblies could exhibit red fluorescence from the mRFP1. Those that pass this round were inoculated in LB medium and the plasmids harvested for sequencing. When a successfully assembled plasmid had been found it was transformed, through electroporation, into *L. lactis* DSM20016 and *E. coli* BL21, after which construct functionality could be tested.

### Primary construct testing

The first round of assemblies involves the insertion of the individual promoters, the TetR inverter, the temperature sensors and the light sensor into pYTK095-based mRFP1 quantification constructs. With these constructs the individual components of the final kill switches could be quantified. So after assembling these plasmids we measured for red fluorescence at various temperatures.

To do this the bacteria containing the constructs were first grown overnight in LB medium with ampicillin, then diluted 1:200 into new LB medium with ampicillin to achieve exponential growth. These cultures were diluted to OD600 0.4-0.6±0.3 in LB- or M9 medium with ampicillin and pipetted in triplicate into a 96-well microtiter plate. These plates were incubated in temperature-controlled microtiter plate readers at various temperatures. Though initially the desire was to test at 26℃, 30℃, 35℃, 37℃, 39℃, 41℃ and 43℃, only 4 experiments could be performed in time: 26℃, 30℃, 37℃ and 41℃. See Results for the data of these experiments.
Had the light sensor been available, a similar experiment would have been developed where the cells would be exposed to different intensities of light at different wave-lengths, to determine sensor sensitivity.

Due to the time constraints and synthesis complications the final killswitch constructs could not be assembled, but had they been constructed a series of growth-curves would have been made at different temperatures, showing whether the temperature kill-switches would inhibit cell-growth, and to what extent. Based on this data a preliminary analysis of kill-switch efficacy could have been performed, which could then have been optimized using promoter exchange, vector variation, and the modification of other conditions.

For the light kill-switch the same could have been done, but using light-exposure instead of variable temperature. Different light intensities and wave-lengths could have been tested to characterize kill-switch sensitivity.

## Temporary deactivation mechanism

Since the killswitch induces cell-death when exposed to light and low temperature, the spray delivery mechanism would likely result in the death of the cells before delivery could be completed. Hence the need for a temporary deactivation mechanism is required. This mechanism is based on the lac repression system, where the lac repressor is capable of binding to a lac repressor binding site on the DNA, preventing transcription from progressing. Cloning this lac repressor between the promoter and the Cas9 of the killswitch would result in the prevention of Cas9 expression when the lac repressor is expressed. Simply controlling lac repressor expression with an inducible promoter like T7 would then allow for tight killswitch control. The inducer simply needs to be added to the liquid containing Nanobuddy, which results in killswitch deactivation, after which the product is sprayed over chickens. Then once the droplets reach a surface the inducer will be diluted over time causing the deactivation mechanism to be turned off.

Deactivation could quite easily be tested with IPTG if the T7 promoter were to be used, however, IPTG cytotoxicity would need to be tested, as well as system leakiness. Due to time constraints this concept is therefore left conceptual for future research. In this research the following constructs are to be developed:

*Figure 38. Temporary deactivation mechanism.*

First, IPTG deactivation of the Lacl repressor is to be tested, requiring knowledge of Placl activity. Hence a Placl-controlled GFP is to be constructed (A). Next, the repressor itself can be tested, where the previously used J23100 promoter (strong) controls Lacl repressor expression, which in turn represses GFP expression. Finally, the deactivation mechanism itself can be developed (B), where the Lacl operator is cloned between the Ptet or PcarH promoter and the Cas9 gene. The inducible promoter and Lacl repressor gene itself is simply placed anywhere on the plasmid.

# Genomic integration

Though plasmids are widely used as carriers for various modified genes and constructs, these require selection pressure to remain functional, often applied using antibiotics and respective antibiotic resistance markers. However, inside the lungs of chickens the application of such antibiotics is unfeasible and unsafe. Hence another method must be used to ensure the retention of desirable genetic material. Genomic integration is the easiest method for which various tools are available. CRISPR-Cas9 combined with HDR (homology directed repair) is the simplest method, however, this requires the in-vivo expression of Cas9 to function, which would activate the killswitch mechanism resulting in cell-death. Therefore, it is recommended to instead use Lambda red recombinase-mediated genomic integration instead, which involves the expression of Lambda red recombinase in the presence of dsDNA with homologous end-regions identical to the recombination target sequence, resulting in site-directed recombination and gene insertion. This method allows for the use of relatively high molecular weight integration cassettes of at least 50 kb, which is ideal for our large constructs [19].

# Engineering cycle

As can be seen in more detail in the Results page, the nanobody expression constructs were tested on expression capacity, but very little of our desired protein was found on the SDS-PAGE gels. We attempted to optimize the expression in various ways, but this did not help much. So in the end we decided to overexpress our nanobodies by replacing the initial IPTG inducible Lac promoter with the more powerful T7 promoter.

To do this we designed only a single primer (p65), targeting the RBS and beginning of the nanobody coding gene, and possessing a long non-binding tail containing the T7 promoter and a BsaI restriction site. As a reverse primer the existing p5 primer was used. When PCR is performed with these primers on pRUC2206-10 and 37, the Lac promoter is excised, and the BsaI restriction site and T7 promoter are added in front of the nanobody gene instead, requiring only Golden Gate Assembly with this one DNA fragment to re-circularize and have the promoter be fully exchanged, forming pRUC22(06-10)-7 and pRUC2237-T7. See figure 39 below for the binding of primer p65, figure 40 for an example of the PCR on pRUC2206, and figure 41 for the overview of the used plasmids and parts.

*Figure 39. Binding of primer p65.*

*Figure 40. Example of the PCR on pRUC2206.*

*Figure 41. Overview of the used plasmids and parts.*

In figure 41 the intermediate linearized fragments have also been marked as individual parts to allow for proper registration in the iGEM registry page.

Due to time constraints, despite attempting, we were not able to successfully assemble and transform the T7 replacement plasmid, however this represents an example of our engineering cycle, as we constructed a plasmid, tested it for activity, then used the collected information to improve the assembly.

# Part improvement

While all the constructs we designed and built in our project were based on existing developed parts and research, one component in particular is of note. The sgRNA part BBa\_K2457002 was designed by iGEM17\_Amazonas\_Brazil to target LacZ, and contains the BBa\_J23100 promoter followed by the optimized sgRNA and the L3S2P21 terminator, all in the standard biobrick format. For our purpose we synthesized the sgRNA without the biobrick prefix and suffix, with a crRNA specific for *L. reuteri* 16S rRNA, and with a BsaI restriction site on either end of the fragment cutting inward, allowing for incorporation into our kill-switches (BBa\_K4233055).

Though adaptation of the Brazilian biobrick to target a different sequence was not difficult, de novo synthesis of dsDNA can become quite expensive if a library of sgRNA is to be generated. To solve this problem we modified the biobrick BBa\_K2457002 by replacing the 20 bp crRNA region with two outward-facing BbsI type-IIS restriction sites and 2 bp of spacers, resulting in the formation of a self-excision module (see figure 42). All that is needed to insert a library is to clone this part into a plasmid of choice, then order two primers containing the crRNA library and overhangs, annealing these primers to form dsDNA with overhangs, and performing a Golden Gate Assembly with the annealed crRNA library and the BBa\_K4233081 part (figure 43).

*Figure 42. A self-excision module.*

*Figure 43. Insertion of a library.*

Due to time constraints this construct has not been cloned, but it has been designed in-silico and uploaded to the registry, allowing for easy synthesis by future iGEM teams.

# References

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[5] Lizier, M. et al. (2010) “Comparison of expression vectors in lactobacillus reuteri  strains,” FEMS Microbiology Letters, 308(1), pp. 8–15. Available at: https://doi.org/10.1111/j.1574-6968.2010.01978.x.

[6] Nucleotide blast: Search nucleotide databases using a nucleotide query (no date) National Center for Biotechnology Information. U.S. National Library of Medicine. Available at: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST\_SPEC=GeoBlast&PAGE\_TYPE=BlastSearch.

[7] Lee, M.E. et al. (2015) “A highly characterized yeast toolkit for modular, Multipart Assembly,” ACS Synthetic Biology, 4(9), pp. 975–986. Available at: https://doi.org/10.1021/sb500366v.

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[9] Kubala, M.H. et al. (2010) “Structural and thermodynamic analysis of the GFP:GFP-Nanobody Complex,” Protein Science, 19(12), pp. 2389–2401. Available at: https://doi.org/10.1002/pro.519.

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[13] Liu, F. et al. (2019) “Biodetoxification of fungal mycotoxins Zearalenone by engineered probiotic bacterium lactobacillus reuteri with surface-displayed lactonohydrolase,” Applied Microbiology and Biotechnology, 103(21-22), pp. 8813–8824. Available at: https://doi.org/10.1007/s00253-019-10153-1.

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[18] Piraner, D.I. et al. (2016) “Tunable thermal bioswitches for in vivo control of Microbial Therapeutics,” Nature Chemical Biology, 13(1), pp. 75–80. Available at: https://doi.org/10.1038/nchembio.2233.

[19] Juhas, M. and Ajioka, J.W. (2016) “Lambda red recombinase-mediated integration of the high molecular weight DNA into the escherichia coli chromosome,” Microbial Cell Factories, 15(1). Available at: https://doi.org/10.1186/s12934-016-0571-y.

[20] Hufton, S. E. er al. (2014) “The Breadth of Cross Sub-Type Neutralisation Activity of a Single Domain Antibody to Influenza Hemagglutinin Can Be Increased by Antibody Valency” PLoS One. 2014; 9(8): e103294. Available at: https://doi.org/10.1186/s12934-016-0571-y.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

[General Experiments](#1)
[Cloning](#2)
[PCR](#3)
[Competent cells](#4)
[Transformation](#5)
[Growth Media](#6)
[Protein Experiments](#7)
[Lung Microbiome DNA Extraction Experiments](#8)
[Primers List](#9)
[References](#references)

# Experiments & Protocols

*"A normal person sees a failed expirement, a scientist sees progress." -Steven Magee*

Hey fellow lab rats!
This page gives an overview of all the different wet lab protocols that our team has utilized during our iGEM project. The page is structured in several sections which indicate the purpose of the associated protocols e.g. SDS PAGE is under Protein experiments since it is only used for proteins. Click on one of the drop down menus and find out all the required reagents and steps for the completion of our experiments.

To get an overview of our laboratory progress head over to the Notebook page. Or if you're more interested in the results see our Results page. Finally to learn more about our engineering approach and design choices head over to the Engineering page.

# General Experiments

## Cloning

For our cloning we initially designed the project to work with Gibson cloning as well as with Golden Gate assembly. The choice for the cloning method was dependent on which primers we used to amplify the fragments that we wanted to incorporate into our desired plasmid.

*Gibson Cloning*

In this protocol,you can find how to perform Gibson cloning.

Materials:

* Gibson master mix.
* Backbone fragment.
* Insert fragment.

Protocol:

1. For Gibson plasmid assembly prepare the following reaction mixture in a 100 µL PCR tube according to figure 1.
![](https://static.igem.wiki/teams/4233/wiki/experiments-1.png)

*Figure 1. Overview of the composition of the Gibson cloning reaction mixture.*

2. Mix gently and incubate at 50°C for 45-60 minutes.

If the Gibson master mix isn’t available, make new according to figure 2.

1. Prepare 5x isothermal reaction buffer. Recipe for 4 mL (don’t combine materials immediately. Read the steps):
2. Mix dNTPs, NAD+, Tris-HCl, MgCl2 and DTT.
3. Slowly add PEG-8000 to the mixture and mix well until completely dissolved. Add H2O to a final volume of 4 mL.
4. Prepare aliquots of the 5x isothermal buffer as required. Store at -20C.
5. Prepare 1,33x Gibson Assembly Mastermix (figure 3). Recipe for 25 x 15 uL aliquots (don’t combine materials immediately. Read the steps):
6. Work on ice. Mix H2O and 5x buffer, then add enzymes.
7. Prepare 25 x 15 uL aliquots in PCR tubes. Store at -20C.

Note: The aliquots are concentrated in 1,33x. Add DNA in a volume of 5 uL to a final volume/concentration of 20 uL/1x.

![](https://static.igem.wiki/teams/4233/wiki/experiments-2.png)

*Figure 2. Overview of the composition of 5x isothermal reaction buffer.*

![](https://static.igem.wiki/teams/4233/wiki/experiments-3.png)

*Figure 3. Overview of the composition of the Gibson master mix.*

*Golden Gate Cloning*

In this protocol, you can find how to perform Golden Gate cloning.

Materials:

* Golden Gate master mix.
* Backbone fragment.
* Insert fragment.
* T4 ligase buffer.
* T4 ligase
* MilliQ

Protocols:

1. For plasmid assembly prepare a reaction mixture in a 100 µL PCR tube according to figure 4.
![](https://static.igem.wiki/teams/4233/wiki/experiments-4.png)

*Figure 4. Overview composition of Golden Gate reaction mixture.*

2. Mix gently by pipetting up and down.
3. Place the tubes in a PCR machine and follow the protocol from figure 5.
![](https://static.igem.wiki/teams/4233/wiki/experiments-5.png)

*Figure 5. PCR protocol Golden Gate reaction.*

## PCR

*Polymerase Chain Reaction*

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

Materials:

* Forward and Reverse primer for your DNA plasmid.
* Phusion buffer (5x).
* dNTPs.
* MilliQ.
* Phusion DNA polymerase.

Protocol:

1. Prepare PCR mixture based on figure 6.
2. The PCR mix is mixed by gently pipetting up and down, followed by insertion into a PCR machine with the program depicted in figure 7.
3. Primer annealing temperature is determined using Snapgene for each primer. Then the found Tms are chosen minus 3-5°C. What amount is subtracted depends on the difference in Tm between the Fw and Rev primers. E.g. a primer of Tm= 61°C and one of Tm= 59°C will be run at an annealing temperature of 56°C.
4. Primer extension depends on the desired length of the fragment, with 30 seconds per kb of product length.

![](https://static.igem.wiki/teams/4233/wiki/experiments-6.png)

*Figure 6. PCR mixture.*

![](https://static.igem.wiki/teams/4233/wiki/experiments-7.png)

*Figure 7. Touchdown PCR program.*

*Colony PCR*

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

Materials:

* Forward & Reverse primer for your DNA plasmid.
* Phire Hot Start II PCR Master Mix.
* MilliQ.

Protocol:

1. Resuspend a colony in 20 μL sterile water.
2. Prepare the PCR mixture by adding together 10 uL of Phire hot start 2x, 1uL forward primer, 1 uL reverse primer, and 7 uL dH2O.
3. To the PCR mixture add 1 uL of the resuspended colony mixture.
4. Primer annealing temperature is determined using Snapgene for each primer. Then the found Tms are chosen minus 3-5°C. What amount is subtracted depends on the difference in Tm between the forward and reverse primers. E.g. a primer of Tm= 61°C and one of Tm= 59°C will be run at an annealing temperature of 56°C.
5. Primer extension depends on the desired length of the fragment, with 15 seconds per kb of product length.

![](https://static.igem.wiki/teams/4233/wiki/experiments-8.png)

*Figure 8. Touchdown PCR protocol for colony PCR.*

*Sequencing*

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

Materials:

* GeneJET Plasmid Midiprep Kit.
* 1,5 ml tubes for each sample respectively.
* Overnight culture of bacteria containing DNA plasmid.

Protocol:

1. Perform Plasmid Midiprep Kit.
2. Perform Nanodrop on obtained samples to determine the DNA concentration.
3. Add 5 µL of plasmid solution to a 1,5 mL tube.
4. Add 5 µL of 10 uM forward & reverse primer, for the plasmid respectively.
5. Label tubes and send them for sequencing.

*Agarose gel electrophoresis*

In this protocol, you can find how to perform an Agarose gel electrophoresis, to separate DNA samples based on their size.

Materials:

* Agarose.
* SERVA DNA Stain G (working dilution is 1:20,000 to 1:50,000).
* TAE buffer.

Protocol:

1. Weigh x% m/v of agarose gel and place this in a flask.

1. When 20 mL of 1% agarose is made, 0.2 grams of agarose is weighed.
2. When 20 mL of 3% agarose is made, 0.6 grams is weighed, etc.

2. Add the TAE buffer and dissolve using a microwave.

1. When microwaving be sure to place a piece of paper (not the writing type, the toilet- or kitchen variant) underneath the flask in case of spilling.
2. Keep an eye on it and don’t let it boil too much.

3. Safety warning: From here on out, equip 2 layers of (blue) gloves and be sure not to touch anything which has come into contact with Ethidium Bromide (EtBr) with bare hands. It’s carcinogenic. Double gloves allow you to remove a layer if you touch any gel/EtBr directly without risking contact.

Note: we used SERVA DNA Stain G, which can also be used in place of EtBr, which is less risky but still caution is advised.

1. Also means that you can only keep anything contaminated with EtBr in the designated area. This includes the gel, the gloves, the pipettes. A special trash-can will be placed nearby for waste.

1. One exception is the gel when you go for viewing, as this is unavoidable. But even then, don’t touch any part of the machine except the glass gel-plate with contaminated gloves. Also clean everything afterwards with paper and water.

2. When you get into direct contact with EtBr, immediately wash thoroughly with water and soap and you should be fine.
3. Gels are poured into a mold followed by the immediate addition of 4 µL of ethidium bromide, which is mixed using the pipet tip as a stirrer.
4. Don’t forget to add a comb.

4. Wait for solidification, which may take around 20 minutes.
5. Place the gel (with the see-through support but without the red barriers) into the Electrophoresis Reservoir.
6. Gently remove the comb and add 1x TAE to the reservoir. The TAE can be re-used a few times, but try to replace it once every day or two to keep the gel free from contamination.
7. Carefully add the sample + dye to the wells.

1. Dye is often 6x concentrated, so for 5 µL samples you’d need to add 1 µL dye, for 20 µL samples you’d need 4 µL dye, etc. Dye can be made by ourselves, or bought, varying the concentrations.
2. The volume per well depends on the comb used, but can be anywhere from max. 15 µL to 50 µL. You could do some testing to find out the maximum.
3. Tip: When you have the sample + dye in your pipet tip, the moment it touches the TAE some of the sample may come out. So penetrate the surface at the bottom deeper part of the reservoir, then move to the wells. This way you don’t accidentally spill any sample in the wrong well.
   Then after gently releasing the sample into the well, move up a bit and suck in 1 µL of the TAE so you don’t drag up the sample while moving out of the liquid again.

8. Close the machine and be sure to connect the wires correctly.

1. The positive electrode pulls on the negatively charged DNA, so have the positive electrode at the bottom, and the negative electrode on the well-side.

9. Run the electrophoresis at 80 V until the dyes have traveled through 80% of the gel, which will take 40-60 minutes. Higher voltages are possible to speed up the process, but this may melt the gel if you do it too much.
10. After electrophoresis, turn off the machine, remove the lid, and carefully lift the gel out of the TAE.

1. The gel is not stuck on the support, so it may float. Be sure you prevent that.

11. Let the liquid seep out and remove the gel from the support with a double-gloved hand.
12. Carefully remove one layer of gloves from one hand, so the newly revealed glove is uncontaminated, and use this hand to open the UV imager machine.

1. Then image the gel, followed by removal of the gel and cleaning of all materials.

## Competent cells

*E.coli DH5a*

In these two protocols, you can find two ways to make competent Escherichia coli DH5𝛂 or E.coli BL21 cell aliquots.

Materials 1:

* LB media.
* Ice-cold 0.1 M CaCl2.
* Ice-cold 0.1 M CaCl2 + 10% glycerol.
* Sterile tubes (1,5 ml).

Protocol 1:

1. Inoculate the E.coli DH5a in 2 mL LB medium for overnight growth at 37℃ and at 220 RPM.
2. After overnight growth, inoculate fresh medium in a sterile erlenmeyer flask with a volume ratio of 1/100. Usually this means 20 mL LB with 0.2 mL of the overnight culture, but it can be scaled up, which is recommended when an -80℃ freezer is available.
3. Grow at 37℃, 220 RPM until an OD of 0.4-0.6. Measure the OD with a spectrophotometer.

1. When the desired OD is approaching, pre-cool a centrifuge.

4. Transfer the culture to a sterile 50 mL tube and spin down in a pre-cooled centrifuge, at 5℃ for 10 minutes at 5000 RPM.
5. Discard the supernatant and add 0.5\*[Culture Volume] of ice-cold 0.1 M CaCl2. Resuspend carefully and incubate on ice for at least 30 minutes.
6. Spin down the cells in a pre-cooled centrifuge at 4℃ , for 10 minutes at 5000 RPM. Discard the supernatant carefully.
7. Resuspend the cells in 0.1\*[Culture Volume] ice-cold 0.1 M CaCl2 + 10% glycerol. Aliquot in 100 µL fractions in sterile 1.5 mL tubes stored at -80 (or -20)℃

Materials 2:

* TYM media:

+ 20 g/L bacto-tryptone.
+ 5 g/L Yeast extract.
+ 0.1 M NaCl.
+ 10 mM MgSO4.

* TfB I:

+ 30 mM KAc.
+ 50 mM MnCl2 (sterilize through filter, add later).
+ 0.1 M KCl.
+ 10 mM CaCl2.
+ 15% glycerol.

* TfB II:

+ 10 mM Na-MOPS, pH 7.0.
+ 75 mM CaCl2.
+ 10 mM KCl.
+ 15% glycerol.

Protocol 2:

1. Inoculate a colony in TYM medium.
2. Grow cells to midlog phase (OD600 = 0,2-0,6).
3. Transfer cells to 100ml of TYM.
4. Grow cells to OD600 = 0,5-0,9.
5. Add TYM medium to 500ml.
6. Grow cells to OD600 = 0,6.
7. Cool cell culture on ice (keep cells cold from now on).
8. Centrifuge 15min, GSA rotor, 4200rpm.
9. Resuspend pellet in 100ml of cold TfB I (on ice).
10. Centrifuge 10min, GSA rotor, 4200rpm.
11. Resuspend pellet in 20ml of cold TfB II (on ice).
12. Aliquot in 300µL (use 150µL per transformation).
13. Freeze in liquid nitrogen.
14. Store at -80°C.

*L. reuteri DMS20016*

In this protocol, you can find how to make competent Lactobacillus reuteri DSM20016 cells for immediate use.

Materials:

* MRS media.
* MRS broth containing 2% glycine and 0.5 M sucrose.
* Cold deionized water.
* 50 mM EDTA (pH 8.0)
* 0.3 M sucrose

Protocol:

1. Grow L. reuteri overnight at 37˚C in MRS broth
2. Inoculate 15 mL MRS broth containing 2% glycine and 0.5 M sucrose with the overnight L. reuteri culture. The glycine acts as a cell wall weakening agent.
3. Grow the cells at 37˚C to an OD600 of 0.2 (~3 hours).
4. Spin down the cells at 6000 g for 10 minutes at 4 ˚C
5. Wash the cells twice with 5 mL cold distilled water
6. Incubate the cells in 50 mM EDTA (pH 8.0) for 10 min
7. Wash the cells with 0.3 M sucrose
8. Resuspend the cells in 100 uL 0.3 M sucrose as electroporation buffer

## Transformation

*E.coli transformation, heat shock*

In this protocol, you can find how to transform competent E.coli cells. (how to insert plasmid DNA into your bacteria).

Materials:

* Ice
* Prewarm heat block or water bath at 42ºC
* Selective plates with the corresponding antibiotics
* Competent cells
* Plasmid DNA

Protocol:

1. Defrost 150 µl aliquot of E. coli competent cells per transformation on ice.
2. Add 1 μl plasmid DNA (10 pg/μl), incubate 15 min on ice.
3. Heat shock the cells for 1 min at 42°C.
4. Immediately place the cells on ice for 2 min.
5. Add 1mL LB and incubate at 37°C for 1 hour (200rpm).
6. Plate out on selective plates.
7. Incubate plates overnight at 37ºC.

*L. reuteri transformation, Electro shock*

In this protocol, you can find how to transform competent L.reuteri cells. (how to insert plasmid DNA into your bacteria).

Materials:

* Recovery medium (sterile, kept at 4°C):

+ M17 (amount dependent on volume).
+ 0.5M sucrose.
+ 0.5% glucose.
+ 20 mM MgCl2 (prepared as stock and sterilized separately).
+ 2 mM CaCl2 (prepared as stock and sterilized separately).

* GM17-agar (sterile and poured in standard petri-dishes with appropriate antibiotics):

+ M17 (amount dependent on volume).
+ 0.5% glucose.
+ 1.5% agar.

Protocol:

1. Thaw competent L. lactis cells on ice.
2. Add a maximum of 10 µL of the DNA to be transformed to the competent cells. Immediately continue to the next step.
3. Transfer the cell/DNA mixture to a sterile electroporation cuvette.
4. Place the cuvette in the electroporation machine and run for approximately 4-5 seconds at the following settings: 2.25 kV, 25 µF and 200 Ω.
5. Add 1 mL of recovery medium to the electroporation cuvettes and carefully mix, then transfer the suspension to 2 mL tubes.
6. Incubate for 2 hours at 30°C.
7. Plate 100 µL of the suspension per plate. It is also possible to then pellet the cultures and resuspend in lower volumes to concentrate the cells, so more colonies could form. This is at risk of covering the plates too much, making differentiation between colonies impossible.

## Growth Media

*Lysogeny Broth (Lennox) media*

In this protocol, you can find how to make LB media.

Materials:

* LB (lennox) powder.
* Deionized water.

Dissolve 20 grams of LB-medium powder in 1 liter of deionized water. Autoclave bottle and close lid when cooled down.

Composition:

* NaCl, 5 g/L.
* Tryptone, 10 g/L.
* Yeast extract, 5 g/L.

*De Man, Rogosa & Sharpe (MRS) media*

In this protocol, you can find how to make MRS media.

Materials:

* MRS (Millipore) powder.
* Deionized water.

Dissolve 51 grams of MRS-medium powder in 1 liter of deionized water. Before autoclaving, preheat the autoclave to limit the glucose from caramelizing. Autoclave bottle and close lid when cooled down.

Composition:

* Peptone 10,0 g/L.
* Meat extract 8,0 g/L.
* Yeast extract 4,0 g/L.
* D(+)-Glucose 20,0 g/L
* Dipotassium hydrogen phosphate 2,0 g/L.
* Sodium acetate trihydrate 5,0 g/L.
* Triammonium citrate 2,0 g/L.
* Magnesium sulfate heptahydrate 0,2 g/L.
* Manganous sulfate tetrahydrate 0,05 g/L.
* Final pH 6.2 +/- 0.2 at 25°C.

*M9 minimal salts media*

In this protocol, you can find how to make M9 media.

Composition for 1 liter:

* 200 mL 5x M9 salts (Sigma Aldrich), autoclaved.
* 2 mL 1M MgSO4 (Thermofisher), autoclaved.
* 100 uL 1M CaCl2 (Thermofisher), autoclaved.
* 780 mL dH2O, autoclaved.
* 20 mL 20% Glucose, filter sterilized (add after autoclaving).

*Plates with media*

For the creation of Agar plates with a certain media, all you have to do is add 15 grams of agar to 1 Liter of media respectively before autoclaving. Keep the media at 60 degrees Celsius after autoclaving to prevent the media from stolling. If you want to add selective antibiotic markers, let the media cool down until you can hold the bottle comfortably for over a minute in a gloveless hand and add the antibiotics before pouring the plates. Use about 15-20 ml per plate.

## Protein Experiments

*ELISA*

In this protocol, you can find how to perform an ELISA experiment.

Materials:

* 96 well plate (maxisorp plates from nunc).
* Desired hemagglutinin proteins.
* PBS.
* PBS with 3% (w/v) skimmed milk powder.
* PBS with 0,05% (v/v) Tween-20.
* Anti c-myc 9E10-HRP.
* OPD.
* 2 M sulphuric acid.

Protocol:

1. Coat the plates with 0.1 mL hemagglutinin at 1 µg/mL overnight in PBS at 4 ˚C.
2. Wash the wells 3 times with 300 µL PBS.
3. Block the wells with 200 µL PBS 3% (w/v) dried milk protein for 1 hour at room temperature.
4. Wash the wells 3 times with 300 µL PBS containing 0.05% (v/v) Tween-20.
5. Add crude extracts (25% v/v) or purified nanobody (100 µL) to the plate and incubate for 1 hour at room temperature.
6. Wash 5 times with 300 µL PBS containing 0.05% (v/v) Tween-20.
7. To detect antibody binding 100 µL of anti c-myc 9E10-HRP (1/1000 dilution) (Roche) in 2%.
8. (w/v) milk powder in PBS was added. Plates were incubated for 1 hour at room temperature.
9. Wash the wells 3 times with 300 µL PBS containing 0.05% (v/v) Tween-20.
10. Add 100 uL chemiluminescent reaction (OPS).
11. Stop the reaction by adding 50 uL 2 M sulphuric acid.
12. Read the plate at 450 nm.

*SDS PAGE*

In this protocol, you can find how to prepare and perform a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) protein separation.

Materials:

* 30% Acrylamide/Bis Solution 37.5:1 (2.6%C).
* TemeDel.
* 10% Ammonium persulfate (APS).
* 25 mM Tris, 192 mM glycine and 0.1% SDS, with pH adjusted to 8.7 (running buffer).
* 25 mM Tris, 192 mM glycine and 0.1% SDS, with pH adjusted to 6.8 (stacking buffer).
* Iso-propanol.

Protocol:

PART I: pouring the gel.

1. Prepare running gel according to figure 9.
2. Pour the gel with a pipet between the glasses for the SDS Page, leave enough room for the stacking gel on top and the comb (which should hover about half a centimeter above the running gel.
3. To remove bubbles and create a flat top of the gel pipet iso-propanol on top of the gel. (discard after the gel has solidified)
4. Prepare stacking gel according to figure 9
5. Pour the gel with a pipet to the top and place the comb for the slots.
6. After the stacking gel has solidified, remove the comb straight and smooth in one go. Then place the gel in the gel electrode assembly with the smaller glass facing inwards.

Part II: running the gel.

7. Fill the inside of the holder to the top with a running buffer and ensure the electrode at the bottom is submerged.
8. Load the samples with the loading dye.
9. Run the gel at 100 volt for about an hour.
10. Stain the gel afterwards with coomassie blue staining for about 20-30 min (this process can be sped up by microwaving the gel shortly)
11. Destain the gel overnight while submerged in deionized water and shaking.

![](https://static.igem.wiki/teams/4233/wiki/experiments-9.png)

*Figure 9. Showing composition of running and stacking gels according to desired percentage of Acrylamide.*

*Western Blotting (through wet-transfer)*

In this protocol, you can find how to perform a Western blot used to visualize certain proteins.

Materials:

* Whatmann papers cut to squares the size of the gel (6 for 1 gel).
* 2 sponges (for 1 gel).
* 100 mL methanol.
* Ice pack filled with frozen blotting buffer.
* PVDF or nitrocellulose membrane for western blot (always wear gloves when handling!).
* 1X PBS:

+ 4 mM KH2PO4.
+ 14 mM Na2HPO4.
+ 115 mM NaCl.

* 1X PBS-T.

+ Add 0.05% Tween20 to PBS

* Fresh 1X blot buffer (2L).

+ 14.4 g Glycine.
+ 3g Tris.
+ Add dH20 up to 850 mL.
+ Add 150 mL methanol.

* Blocking buffer.

+ 0.2% I-block in PBS-T. Warm up PBS and dissolve I-block in there, then add Tween. Tween cannot be heated.

* SuperSignal West Pico Plus Chemiluminescent Substrate.
* Fuji imager.

Protocol:

PART I: Protein transfer to membrane.

1. Incubate a SDS-PAGE gel for 15 min in blot buffer, as well as 3 stacks of Whatmann papers.
2. Incubate the membrane for 15 min in methanol and dip it in blot buffer. *Make sure the membrane does not dry out during assembly of the sandwich!*
3. Assemble the sandwich in the following order in the blotting casette:

1. Black side (-).
2. Sponge
3. 3x Whatmann paper.
4. SDS-PAGE gel.
5. Membrane.
6. 3x Whatmann paper.
7. Sponge
8. Red/White side (+).

4. Remove air bubbles by rolling over the sandwich (i.e. with an empty falcon tube).
5. Close the sandwich and transfer to a running tank, black on the black side. Add blot buffer and an ice pack (filled with frozen blotting buffer).
6. Run for 1.5h at 100V.
7. Carefully take out your membrane and put it in a 50ml falcon tube using tweezers (make sure it does not dry out!). The protein side of the membrane should be facing to the inside of the tube.
8. Swiftly fill the falcon tube with blocking buffer.
9. Block your membrane overnight in the cold room, or for 1h at RT. Remember that the falcon tube should always be shaking/turning.

PART II: Visualization

1. Discard the blocking buffer buffer and shortly rinse the membrane with PBS-T in a squared petri dish.
2. Add 1:10.000 antibody (anti-his-HRP) diluted in 0.1% I-block in PBS-T (15 mL for one blot in a square petri dish) and incubate for 1h shaking at RT.
3. Collect the antibody and store it in -20C, it can be used several times.
4. Wash the membrane 3x 10 min shaking at RT with PBS-T.
5. Prepare 1mL of SuperSignal West Pico Plus Chemiluminescent Substrate (500ul of each component).
6. Take the membrane out of the PBS-T, drain of excess liquid and transfer the membrane into a plastic sheet. Add the 1mL of substrate on top of the membrane.
7. Image the blot in the Fuji imager by chemiluminescence/increments on a black background and -without moving the blot- also take an image of the protein ladder in the EPI channel.

*Sonication*

In this protocol, you can find how to perform Sonification used to break cells apart.

Materials:

* Sonicator.
* Lysis buffer.
* Ice.

This protocol begins when a culture of cells is available.

1. Centrifuge cells to pellet them (5000 RPM for 5-10 minutes for moderate pelleting). Discard the supernatant (or in the case of a protein secretion experiment, save the supernatant for further testing).
2. Resuspend the cells in a lysis buffer.

1. Usually contains PMSF (phenylmethylsulfonyl fluoride) which prevents protein degradation.

3. Chill the cell solution in ice, and keep the sample in ice throughout the process.

1. Sonication generates heat which might damage proteins.

4. Put the sonication probe inside the tube with the chilled resuspended cells.

1. Make sure the bottom of the probe is fully submerged, but don’t let it touch the bottom of the tube.
2. Keep an eye on the sonication, as foaming indicates protein denaturing.

5. Determine the parameters of the sonication.

1. Frequency: Often set between 20-50 kHz, depending on the lysis difficulty and protein sensitivity.
2. Sonication duration varies depending on optimization, but is often from 10-30 seconds.
3. Duration of intervals varies, but is important for cooling down between steps. Can be from 10-60 seconds.
4. One protocol suggests: Sonication until 400J using 50% amplitude and pulses of 10s: 10s (so 10 second duration with 10 second intervals).

*NiNTA His-Tag purification*

In this protocol, you can find how to perform a His-tag purification.

Materials:

* Ni-NTA sepharose resin (beads).
* 1x phosphate buffered saline (PBS).
* 2M imidazole.

Protocol:

1. Add 1 mL Ni-NTA sepharose resin to the column (0.5 mL column volume).
2. Wash beads with 10 CV deionized water.
3. Wash beads with 10 CV 1x PBS + 10 mM imidazole.
4. Add the washed beads to the cell free extract containing the protein of interest. Incubate the cell free extract with the Ni-NTA beads for 1 hour at 4 °C under constant nutation.
5. Add protein & Ni-NTA beads mixture to the column to establish the column. Collect a sample for the flowthrough.
6. Wash the column with 20CV PBS + 50mM of imidazole. Collect a sample from the washing step.
7. Elute the column with PBS + 500mM imidazole. Collect 15 fractions of 150 µl each.

*Bradford assay*

In this protocol, you can find how to perform a Bradford assay.

Materials:

* Coomassie brilliant blue G-250 dye.
* 85% phosphoric acid.
* 95% ethanol.
* Sterile MilliQ.
* BSA.

Protocol:

Below is the GoldBio protein quantification assay.

Bradford reagent preparation:

1. Weigh 100 mg coomassie brilliant blue G-250 dye.
2. Add 50 mL 95% ethanol.
3. Slowly and carefully add 100 mL 85% phosphoric acid.
4. Mix until the blue dye is completely dissolved.
5. Add the dye/ethanol/phosphoric acid solution to 850 mL sterile MQ H2O.
6. Filter any precipitates.
7. Store Bradford Reagent at 4℃ for months.

Measuring Protein Standard:

1. Warm up the spectrophotometer.
2. Pipet 6 different volumes of 0.5 mg/mL BSA into separate cuvettes.

1. E.g.: 0 µL (0 µg), 10 µL (5 µg), 20 µL (10 µg), 30 µL (15 µg), 40 µL (20 µg), 50 µL (25 µg).
2. Other protein standards can be substituted for BSA.
3. The cuvette with no protein standard in it serves as a blank.

3. Add 1.5 mL of Bradford Reagent to each cuvette.
4. Cover the cuvettes with plastic paraffin film and mix by gently inverting.
5. Let the cuvettes incubate at room temperature for 10 minutes.
6. Measure the absorbance of each cuvette at 595 nm.

Measuring the sample of unknown quantity:

1. Pipet between 10 to 50 µL of the protein that is to be quantified into a cuvette; a dilution may be necessary.

1. The goal is to get an absorbance reading that is in the middle of the standard curve.

2. Add 1.5 mL of Bradford Reagent to the cuvette.
3. Cover the cuvette with plastic paraffin film and mix gently by inverting.
4. Let the cuvettes incubate at room temperature for 10 minutes.
5. Measure the absorbance of each cuvette at 595 nm.

1. If the absorbance doesn’t fall within the range of the standard curve, change the dilution of the sample and measure the absorbance again.

Standard Curve Generation:

1. Make a scatter plot for the standard curve values.
2. Plot the micrograms (µg) of standard protein that was assay on the x-axis. Plot the Abs595 of the measurements on the y-axis.
3. Fit a linear trendline to the graph.
4. Show the linear regression equation.
5. Calculate the unknown amount of protein by using the linear regression equation, as shown in the below picture (figure 10):
![](https://static.igem.wiki/teams/4233/wiki/experiments-10.png)

*Figure 10. Formula used to calculate the unknown amount of protein: the linear regression equation.*

# Lung Microbiome DNA Extraction Experiments

*Protocol 1, using homogenization*

This is the DNA extraction protocol we initially used for gathering the DNA from the lungs. It is based on the homogenization strategy of a tissue sample described in [1]. Keep in mind that for analysis of microbiomes without many microbes living in it (e.g. lungs), your microbial DNA concentration might end up too low for further downstream analysis. For this we recommend our second DNA extraction protocol from a tissue sample (not suitable for studying percentages in microbial compositions, only for the presence of certain microbes).

Materials:

* Chicken lungs (acquired as a by-product from the meat industry)
* Liquid nitrogen
* Mortar and pestle
* Biosafety cabinet
* DNeasy PowerSoil Pro Kit

Protocol: an overview of the steps taken can be found in figure 10.

* Lungs (inside 50 mL falcon tubes) were placed in the freezer upon the moment of homogenization.
* Cut frozen lungs into small pieces (approximately 1 cm cubed).
* Homogenize your sample in a biosafety cabinet by grinding with a mortar and pestle to achieve a powdery consistency. Use liquid nitrogen to keep the samples frozen while being homogenized. Tip: separate your cubed sample before adding liquid nitrogen to prevent them from sealing together when freezing.
* Place portions of the tissue homogenates (0.25 g) inside sterile 2.0-ml screw-cap tubes for DNA extraction.
* Extract DNA from the tissue homogenates using a DNeasy PowerSoil kit (Qiagen Sciences Inc., Germantown, MD) according to the manufacturer’s protocol. For the last step, use 150 ul of elution buffer.
* Store extracted DNA at -20ºC before quality control.
* Use a nanodrop to determine the concentration of your DNA.
* Carry out a PCR using V3 / V4 primers to test whether there is bacterial DNA that can be amplified. For a detailed description, check out the PCR quality control protocol below.
* Send DNA samples (on dry ice) for 16S rRNA NGS after confirming that microbial DNA can be amplified.

![](https://static.igem.wiki/teams/4233/wiki/experiments-11.png)

*Figure 11. Overview of the steps taken in the DNA extraction described in the ‘Microbiome DNA extraction protocol (homogenization)’.*

*Protocol 2, using growth chamber*

Due to time constraints we were not able to optimize our previous protocol. In order to still gain results in time for the deadline, we used the following protocol to amplify bacteria in our lung sample using a growth chamber. We added a growth medium to our lung samples and incubated them in order to increase the number of bacteria present. Medium was extracted and analyzed further. This technique is only suitable to identify specific species in your sample, but the NGS cannot say anything about bacterial composition in percentages as you used a growth medium to amplify the number of bacteria in your sample.

Materials:

* Chicken lungs (acquired as a by-product from the meat industry).
* LB medium (steps to make this are described in growth media protocols).
* Biosafety cabinet.
* DNeasy PowerSoil Pro Kit

Protocol:

* Lungs (inside 50 mL falcon tubes) were placed in the freezer upon the moment of adding growth medium.
* Add 20 mL of LB medium to the falcon tubes containing the lungs. Don’t forget to create a negative control using the same medium.
* Thoroughly thaw the sample, and vortex for 2 minutes at max speed.
* Place the samples in an incubator at 37ºC and grow overnight.
* Vortex tubes for 2 minutes on max speed.
* Take out 250 ul of medium containing all of the grown bacteria present in the lung. If your sample somehow does not appear to have grown, put the tube back in the incubator overnight.
* Add the 250ul of medium containing the bacteria inside sterile 2.0-ml screw-cap tubes for DNA extraction.
* Extract DNA from the tissue homogenates using a DNeasy PowerSoil kit (Qiagen Sciences Inc., Germantown, MD) according to the manufacturer’s protocol. For the last step, use 150 ul of elution buffer.
* Store extracted DNA at -20ºC before quality control.
* Use a nanodrop to determine the concentration of your DNA.
* Carry out a PCR using V3 / V4 primers to test whether there is bacterial DNA that can be amplified. For a detailed description, check out the PCR quality control protocol below.
* Send DNA samples (on dry ice) for 16S rRNA NGS after confirming that microbial DNA can be amplified.

*Protocol 3, Quality control through PCR*

This protocol shows how you can use V3 / V4 primers from the hypervariable region of the 16S rRNA to check whether you have bacterial DNA in your sample that can be amplified.

Materials:

* DNA sample.
* Positive control (any bacterial DNA sample).
* Primers: target region V3 / V4 region 16S rRNA.

+ Forward primer 338F sequences: 5'- ACTCCTACGGGAGGCAGCA-3'.
+ Reverse primer 806R sequences: 5'- GGACTACHVGGGTWTCTAAT-3’.

* Phire Tissue Direct PCR Master Mix (Thermo Scientific).

Protocol:

* Prepare primers according to manufacturer instructions.
* Prepare PCR mixtures (total 10 ul):

+ 5 ul phire tissue direct PCR master mix.
+ 1 ul template DNA (your sample).
+ 1 ul forward primer.
+ 1 ul reverse primer.
+ 2 ul MilliQ.

* Start a PCR on the following settings: one cycle of 95°C for 5 min, followed by 35 cycles of 98°C for 20 s, 50°C for 15 s, and 72°C for 1 min.
* Visualize PCR products using the agarose gel electrophoresis protocol described earlier.
* Identify whether you find bands around the 450 bp mark (expected size for the V3/V4 region).

# Primers List

*Primers*

![](https://static.igem.wiki/teams/4233/wiki/primer-list.png)

# References

[1] Ngunjiri, J. M., Taylor, K. J. M., Abundo, M. C., Jang, H., Elaish, M., KC, M., Ghorbani, A., Wijeratne, S., Weber, B. P., Johnson, T. J. & Lee, C. W. (2019, mei). Farm Stage, Bird Age, and Body Site Dominantly Affect the Quantity, Taxonomic Composition, and Dynamics of Respiratory and Gut Microbiota of Commercial Layer Chickens. Applied and Environmental Microbiology, 85(9). https://doi.org/10.1128/aem.03137-18

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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[Introduction](#1)
[Microbiome analysis](#2)
[Nanobody secretion](#3)
[Nanobody efficacy](#4)
[Biocontainment](#5)
[References](#references)

# Results

*"If I have seen further than others, it is by standing upon the shoulders of giants.” - Isaac Newton*

# Introduction

The support for our project stands not on the shoulders of giants but rests on the four pillars that we have defined as follows; Microbiome analysis, Nanobody secretion, Nanobody efficacy, and Biocontainment.

# Microbiome analysis

## DNA Extraction

To test whether lung samples from Dutch poultry actually contained the Limosilactobacillus reuteri strains, we conducted a lung microbiome analysis using lung animal-by-products from Dutch poultry. At first, we used a DNA extraction protocol which relied on the homogenization of our lung tissue before extraction. After numerous troubleshooting steps and optimization, we concluded that the chicken DNA present in the sample was present in significantly different higher amounts, which would block efficient primer binding in our quality control PCR resulting in no bands. Subsequently, we hypothesized that diluting the sample would make the concentration of microbial DNA so low, that the PCR would also not work.

We continued with an alternative protocol, such that we would still be able to gain insights regarding the microbes that live in the poultry lung microbiome. The alternative protocol we developed relied on growing our lung samples in LB medium overnight, meaning we would still be able to identify which microbes are present in the chicken lungs, but we would not be able to say anything about composition ratios.

For the purposes of our research (identifying presence, rather than total composition) these results would still be valuable. We conducted our second DNA extraction protocol (growth based) for 9 lung samples and one negative control (LB medium), and followed up with a quality control of our samples as described in our optimized quality control PCR for microbiome analysis found on our [experiments and protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page under microbiome analysis protocols.

Figure 1 below shows clear bands for all samples around the 450bp mark as anticipated. We used primers for the hypervariable V3/V4 region of microbial 16S rRNA, indicating that our DNA extraction samples contained DNA that could be amplified using these primers. This indicates that our microbiome DNA extraction was successful, and we have extracted microbial DNA which could be applied for downstream sequencing. However, since our negative control also showed a band at the same size, we suspected that the LB medium we had used was contaminated with microbial DNA. To exclude that our samples might have been contaminated during DNA extraction, we grew the remaining, theoretically sterile, LB medium at 37 ℃ overnight. After noticing growth we concluded that our LB was indeed contaminated. This could suggest that the bands seen for each sample is a result of contamination. Even though contamination was observed, we still considered sequencing the samples due to time constraints.

![](https://static.igem.wiki/teams/4233/wiki/results-1.png)

*Figure 1. Quality control for microbial DNA extracts from the chicken lung samples acquired through growing the lungs in LB medium overnight. 1,6% (m/v) agarose gel showing the V3/V4 hypervariable 16S rRNA region (~450bp) amplified PCR products of our DNA extraction samples.*

Figure 2 below shows the nanodrop results we acquired from the aforementioned microbial DNA extracts, showing widely varying purities and final DNA concentrations. Our negative control appears to have very low concentrations of DNA present, indicating that there must have been other bacteria in our lung samples resulting in the increased concentrations. Pressed for time, we decided to continue to Next Generation Sequencing analysis using the microbial DNA extracts, as these results could still confirm the presence of L. reuteri in our samples. As seen in figure 2, we had obtained a high enough concentration (>5 ng/μL per sample), enough DNA in our total sample (>100ng), and had very clear bands around the 450 bp mark on our 1,6% (m/v) agarose gel (figure 1), we concluded that these samples would be sufficient to send for downstream 16S rRNA Next Generation Sequencing to identify whether we could identify L. reuteri in our lung samples.

![](https://static.igem.wiki/teams/4233/wiki/results-2.png)

*Figure 2. Quality control for microbial DNA extracts from the chicken lung samples acquired through growing the lungs in LB medium overnight. Results show the concentration of DNA extracted in the nine lung samples + negative control. Also the 260/280 and 260/230 absorbance ratios are provided as indication of sample purity.*

## Next Generation Sequencing:

We sent our DNA samples for 16S rRNA Next Generation Sequencing (Illumina NovaSeq6000) to a third party (Biomarker Technologies (BMK) GmbH, Münster, Germany). As of now, we are still waiting for the results which we should get any day now. We are looking forward to presenting them at the Grand Jamboree!

# Nanobody secretion

Within the nanobody secretion pillar we focused on the ability of L. reuteri to produce and secrete nanobodies. With this pillar we will show one element of our proof of concept, which is to have L. reuteri produce and secrete nanobodies.

## Growing L. reuteri under anaerobic conditions

When we received L. reuteri DSM 20016 we decided to make initial growth curves at 37 °C in a 96 wells plate, such that we can more easily predict growth over time for future experiments. However, when growing L. reuteri under aerobic conditions little to no growth was observed. This meant that, since L. reuteri is a facultative anaerobic organism, we first had to develop a method to reliably grow L. reuteri in our lab. This was necessary because we did not have an anaerobic chamber.

We developed a method using mineral oil on top of our L. reuteri cell cultures in order to slow down the diffusion of oxygen into the MRS medium to levels the cells find acceptable. When growing the cells with mineral oil on top, as shown in figure 3, more standard growth curves were observed. These growth curves show that we are now able to reliably grow L. reuteri in our lab, using a simple and inexpensive method. From the growth curves it can be observed that growing from an overnight culture the cells reach the exponential growth phase relatively faster (after ~3-7 hours) compared to growing L. reuteri from a glycerol stock (after ~13 hours).

![](https://static.igem.wiki/teams/4233/wiki/figure-3fancy.svg)

*Figure 3. Growth curves of L. reuteri DSM 20016 at 37 °C in anaerobic conditions by adding mineral oil on top of the cell culture. L. reuteri cells were grown overnight at 37 °C in MRS medium. From the overnight culture cells were diluted 10 times (yellow), 50 times (red), 100 times (brown), 200 times (green), and 20 times from a glycerol stock (blue). The diluted cells were placed in a 96 wells plate, to which mineral oil was added on top. The 96 wells plate was kept in the plate reader at 37 °C, where it was read every 5 minutes at 600 nm.*

## Transforming L. reuteri through electroporation

Our next challenge was to transform L. reuteri DSM 20016 by means of electroporation. Being able to insert a plasmid into L. reuteri is a foundational step in our proof of concept regarding the nanobody secretion pillar. Literature has shown DSM 20016 is able to be transformed, which prompted us to reproduce the protocols. However, these initial attempts showed no colonies, bringing us back to the bread and butter of science; optimization. The most important alteration to our electroporation protocol was to include 2% glycine to the cell growth medium. Glycine is a widely used cell wall weakening agent for the transformation of gram positive bacteria.

The results in figure 4 show a band at D1 at the expected size ~1200 bp. Here, the pYTK001 vector containing GFP was used for transformation. The cells were electroporated at 2,0 kV with all other variables as described in our protocol. The vector itself was plasmid purified from E. coli JH101 in order to combat DNA methylation incompatibilities between E. coli and L. reuteri, as hypothesized by the iGEM Oxford 2019 team. Due to time constraints we were not able to investigate further, although the presence of a band at the expected size gives a strong indication that we were able to transform L. reuteri DSM 20016, albeit with a low transformation efficiency.

![](https://static.igem.wiki/teams/4233/wiki/results-4.png)

*Figure 4. Colony PCR of L. reuteri transformed under varying electroporation conditions. Cells were grown at 37 °C to OD600 ≈ 0,7 in MRS medium supplemented with 2% glycine and 0,5 M sucrose. Cell cultures were concentrated 100 times in 30% PEG 600 as electroporation medium. To the concentrated cells 250-500 ng of plasmid DNA was added, after which the cells were electroporated at either 2,0 or 2,5 kV. The electroporated cells were immediately incubated in MRS medium for 4 hours at 37 °C. After incubation the cells were plated with 10 μg/mL of the appropriate antibiotic. Resulting colonies were subjected to colony PCR, where samples A1,A2, and C1-D3 were transformed with the pYTK001 vector containing GFP (~1200 bp expected size) and B1-B3 were transformed with the pTRKh2 vector containing the R1A-A5 nanobody (~1900 expected size).*

Cells were grown at 37 °C to OD600 ≈ 0,7 in MRS medium supplemented with 2% glycine and 0,5 M sucrose. Cell cultures were concentrated 100 times in 30% PEG 600 as electroporation medium. To the concentrated cells 250-500 ng of plasmid DNA was added, after which the cells were electroporated at either 2,0 or 2,5 kV. The electroporated cells were immediately incubated in MRS medium for 4 hours at 37 °C. After incubation the cells were plated with 10 μg/mL of the appropriate antibiotic.

Resulting colonies were subjected to colony PCR, where samples A1,A2, and C1-D3 were transformed with the pYTK001 vector containing GFP (~1200 bp expected size) and B1-B3 were transformed with the pTRKh2 vector containing the R1A-A5 nanobody (~1900 expected size).

# Nanobody efficacy

Within the nanobody efficacy pillar we focused mainly on producing and purifying the anti-avian influenza nanobodies as described in literature [1]. We constructed several monovalent and bivalent nanobodies, which we then cloned into the pTRKh2 expression vector and transformed into E. coli BL21 cells.

## Production and purification of anti-avian influenza nanobodies

In total, we managed to construct four different nanobodies, which we could produce in either E. coli DH5⍺ or BL21. The monovalent R1A-A5 and R1A-B6 were successfully cloned into E. coli BL21, while the bivalent R1A-B6 and monovalent R1A-B6 linked to monovalent R1A-A5 were cloned into E. coli DH5⍺; due to time constraints the two bivalent nanobodies were not (successfully) transformed into E. coli BL21.

The transformed cells were grown at 37 °C in 1 L LB medium to an OD600 = 0,5 ± 0,2, after which the cultures were supplemented with 1 mM IPTG as protein expression inducer. The induced cultures were incubated at 30 °C overnight, followed by sonication and His-tag purification with Ni-NTA agarose. The His-tag purification elution fractions were used for Western blot analysis to prove if we can produce the nanobodies of interest. The results in figure 5 show a clear presence of his-tagged proteins at the expected molecular weights in a broad range of elution fractions for all four nanobodies; this is with the exception of the bivalent R1A-B6 nanobody (figure 5D), where we expect a 30,79 kDa band, yet observe a band at approximately half of that. In the wash out fraction it can be seen that we lose some amount of nanobodies during the purification, thus lowering our overall nanobody production yield.

![](https://static.igem.wiki/teams/4233/wiki/results-5-new.png)

*Figure 5. Western blot analysis of our nanobodies. (A) R1A-B6 monovalent nanobody with an expected molecular weight of 15,46 kDa. (B) R1A-A5 monovalent nanobody with an expected molecular weight of 15,13 kDa. (C) Bivalent nanobody consisting of R1A-B6 linked (head-to-tail) to R1A-A5 using a (gly-gly-gly-gly-ser)6 linker; expected molecular weight of 30,13 kDa. (D) Bivalent nanobody consisting of two monovalent R1A-B6 nanobodies linked (head-to-tail) together by a (gly-gly-gly-gly-ser)6 linker; expected molecular weight of 30,79 kDa. To detect the nanobodies we used an anti-6xhis antibody with an HRP conjugate. For all four blots a 70 kDa polyhis-tagged protein was used as a positive control.*

## Antigen-specific reactivity of anti-avian influenza nanobodies to hemagglutinin with ELISA

The purification of our nanobodies from the cell free extracts by means of his-tag purification proved to be a challenge. Because our nanobody yield is relatively low there is binding competition to the Ni-NTA agarose, significantly reducing the purity of our nanobodies. Since we were unable to successfully purify the nanobodies we decided to pool the elution fractions and use this for our nanobody efficacy assays.

To investigate the antigen specificity of the monovalent and bivalent nanobodies we decided to perform an ELISA experiment on almost the complete range of known influenza subtypes, meaning we went beyond the scope of previous literature. We ordered the hemagglutinin proteins listed in table 1 from BEI resources, which we received as his-tagged protein solutions. Because of the presence of this his-tag on our hemagglutinin antigens, we were not able to use our anti-6xhis HRP conjugate antibody as used previously in our Western blot results. For this reason we opted to use an anti-myc HRP conjugate antibody, which will bind to the C-terminal myc-tag present in all our nanobodies.

*Table 1. Scope of avian influenza hemagglutinins used for ELISA experiments. The range encompasses H1 to H11 with the exception of H6 and H8 subtypes. All hemagglutinin proteins originate from an influenza A virus. Some hemagglutinin proteins originate from a pdm09 strain, which stands for “Pandemic Disease Mexico 2009”, colloquially known as swine flu.*

![](https://static.igem.wiki/teams/4233/wiki/results-6.png)

The ELISA results, as displayed in figure 6, show little to no binding to any hemagglutinin, even to the ones who have shown to bind in literature. The absorbance values we found are significantly lower compared to typical binding values found in literature. Considering this low absorbance is also true for our positive control, it is most likely a technical error somewhere in our ELISA protocol. To optimize our ELISA protocol to scientific standards is beyond the scope of our iGEM timeframe. For future ELISA optimization experiments we are looking at increasing the concentration of hemagglutinin on the 96 wells plate. On top of this, we aim to coat the hemagglutinin on the 96 wells plate in a bicarbonate buffer, which could, due to the buffers higher pH, increase the rate of hydrophobic interactions between amino acid side chains and the plastic surface of the plate [2]. Furthermore, the nanobody production yield requires further optimization, such that protein purification becomes more reliable. As of now, we are aiming to implement the suggested optimizations and we are looking forward to presenting them at the Grand Jamboree!

![](https://static.igem.wiki/teams/4233/wiki/results-7.png)

*Figure 6. Anti-avian influenza nanobody reactivity to a wide range of avian influenza hemagglutinins. Each hemagglutinin subtype was coated on the 96 wells plate at 1 μg/mL in PBS overnight at 4 °C. The coated wells were subjected to the pooled elution fractions of his-tag purified nanobodies. Nanobody binding to hemagglutinin was measured at an absorbance of 450 nm using Benzene-1,2-diamine (OPD) as chemiluminescent substrate.
For the positive control a well was coated with H7N9 hemagglutinin, which was subjected to rabbit serum against H7N9 (BEI resources NR-48765). Then a secondary anti-rabbit alkaline phosphatase conjugate antibody was used to measure the binding. For the negative control an H7N9 coated well was subjected to PBS instead of a nanobody solution. The monovalent R1A-B6 and R1A-A5 nanobodies are shown in grey and yellow, respectively, while the bivalent R1A-B6 and R1A-B6 linked with R1A-A5 are shown in orange and blue, respectively.*

# Biocontainment

The Nanobuddy project involves the development of a therapeutic L. reuteri which can be seeded into the lung mucus of chickens. Such an in situ deployment of a bacterium requires tight biocontainment, ensuring that none of the bacteria escape into the environment. We thus developed a 2-input OR-gated killswitch, designed to be capable of activating a CRISPR-Cas9 kill-mechanism. The kill mechanism gets activated when either the temperature drops below a set number or when there is exposure to light. The specific design-choices and assembly strategy can be found on the [engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) page.

For brevity we will not show all the steps and results from the cloning of all the constructs, but in the end we successfully assembled the plasmids for the testing of individual promoters, the TetR inverter, and the temperature sensors.

## Temperature sensor testing

As outlined in the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) and the [engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) pages, we performed 96-well microtiter plate-based growth and fluorescence experiments on the successfully constructed strains. Here we pipetted 200 µL of our strains with a dilution of approximately 0.01 OD600 into M9 minimal medium with glucose and ampicillin, into the 96-well plate. These plates were then incubated in a TECAN-100 -or a SYNERGY HTX MM microtiter plate reader.

The gain settings of the plate reader were optimized and set to 100. The runtime was optimized and set to 10 hours and measurement frequency was set to every 2.5 minutes. As optimization was time consuming, each measurement had slightly different settings and the results yielded are not very comparable. However, they do indicate some trend and functionality of the constructs as can be seen below.

The first construct of interest is BBa\_K4233041, where the J23100 promoter controls mRFP1. This construct was transformed into DH5α and BL21, and tested on fluorescence over time. The fluorescence was first normalized to the M9 blank, followed by division by OD600 per time-point to determine fluorescence/OD600. The result is shown below in figure 7.

![](https://static.igem.wiki/teams/4233/wiki/results-7-new.svg)

*Figure 7. Normalized fluorescence (575/620) / OD over time (hours) of the BBa\_4233041 construct, consisting of a J23100 constitutive promoter powering mRFP1, in BL21.*

These results (gain: 255, em/ex: 575/620, runtime: 5 hours, measurement frequency:3 min.) show that there are high levels of fluorescence from this construct quite consistently, with an initial drop possibly due to the organism’s fluorescence level adapting to the lower temperature after growth at 37℃. As the mRFP1 slowly degrades older proteins. This data is quite promising for an initial experiment, however, as it shows the construct is functional with relatively consistent expression. Though it also appears that the temperature also significantly affects expression here. We recommend future experiments with more temperatures, and to compare BL21 with DH5α, where consistent run-times, measurement frequency and other parameters are used.

The same experiment was also performed using other parameters (gain: 200, em/ex:575/620, runtime:5 hours, measurement frequency: 2.5 min) and at 37℃. The construct here was only available in DH5α, which exhibited low growth, which caused normalization to drop the relative fluorescence to below 0. This prevents direct quantitative comparison with the previous dataset. However, the general trend is visible, which shows an increase in fluorescence per cell as is expected unlike the previous results. This can be explained by the fact that the initial growth temperature is the same as the experimental temperature, so the cells had already been expressing at the correct temperature.

![](https://static.igem.wiki/teams/4233/wiki/results-8-new.svg)

*Figure 8. Normalized fluorescence (575/620) / OD over time (hours) of the BBa\_K4233041 construct, consisting of a J23100 constitutive promoter powering mRFP1, in DH5α.*

Finally, two of our temperature sensors were also tested, pTlpA1-36C (BBa\_K4233045) and pTlpA2-36C (BBa\_K4233049). These were almost identical, except with differently designed promoters (Mk1, the original from the paper and Mk2, see the [engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) page), the latter of which was hypothesized to function more effectively. In this experiment the parameters were (gain: 200, em/ex: 575/620, runtime: 5 hours, measurement frequency:2.5 min) at 37℃. The constructs were transformed in DH5α, and the results are shown below in figure 9.

![](https://static.igem.wiki/teams/4233/wiki/results-10-new.svg)

*Figure 9. Normalized fluorescence (575/620) / OD over time (hours) of the BBa\_K4233045 and BBa\_K4233045 constructs, consisting of TlpA-based temperature sensor mechanisms (Mk.1 and Mk.2 respectively) powering mRFP1, in DH5α.*

This data appears to have the same issue as the previous graph, where the low cell-growth and the relatively strong M9 background fluorescence causes the normalized fluorescence to drop below 0. The same recommendation repeats here, to optimize cell-growth. Additionally it is possible to use a standard, like a constitutive promoter construct used on every plate to determine fold-change compared to that standard, or to do the same with a serial dilution of the Interlab fluorescent dyes. This would allow for results to be compared more effectively between different plates.

As for this dataset on its own, it appears that maximum promoter activity is the same for both promoter variants, though the original pTlpA1 promoter speeds up more quickly.

For the temperature sensor mechanism itself, since the temperature is only one degree above the inhibition limit, this data is not reliable. But it seems like the repression is not present at this temperature at the very least. To make a conclusion about TlpA repression and temperature sensing, more temperatures above and below the repression limit need to be tested.

# References

[1] Hufton SE, Risley P, Ball CR, Major D, Engelhardt OG, Poole S. The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One. 2014 Aug 1;9(8):e103294. doi: 10.1371/journal.pone.0103294. PMID: 25084445; PMCID: PMC4118869.

[2] What is the difference between bicarbonate and PBS as a coating buffer in ELISA assay? AAT Bioquest, https://www.aatbio.com/resources/faq-frequently-asked-questions/What-is-the-difference-between-bicarbonate-and-PBS-as-a-coating-buffer-in-ELISA-assay

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