iGEM 2022 — Team sydney-australia

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We've been working hard inside and outside of the lab for around four
months, and whilst it has been challenging, we've had a lot of fun! Read
more about it below.

27/6/2022 - 3/7/2022

##### Nanobody surface display

* Parts design of nanobodies and Neae

##### fuGFP-CBD

* Parts design of fuGFP-CBD

##### HP and Modelling

* Brainstormed session of what we would like to do for the Tahgara
  Winter programme
* Delegated tasks and set deadlines to different members

4/7/2022 - 10/7/2022

##### Nanobody surface display

* Ordered parts and associated primers

##### fuGFP-CBD

* Ordered parts and associated primers

##### HP and Modelling

* Prepared materials for the
  [Tahgara Winter programme](communication#tahgaraedu)
* Modelling started in OpenMM

11/7/2022 - 17/7/2022

##### Nanobody surface display

* -

##### fuGFP-CBD

* -

##### HP and Modelling

* Had a practice run and ran the
  [Tahgara Winter programme](communication#tahgaraedu)
  at our lab
* The structure of nanobody H and sfGFP successfully modelled in
  OpenMM

18/7/2022 - 24/7/2022

##### Nanobody surface display

* gBlocks of parts arrive

##### fuGFP-CBD

* gBlocks of parts arrive

##### HP and Modelling

* -

25/7/2022 - 31/7/2022

##### Nanobody surface display

* Inserted Neae surface display construct into TOP10
  *E. coli* and induced with X-gal. Notably, there were few
  blue colonies
* Performed colony PCR on 2 colonies with Neae
* Troubleshot lac expression since there were few blue colonies.
  Maybe due to low copy number plasmid

##### fuGFP-CBD

* Inoculated plates with transformed TOP10 cells with the 4
  fuGFP-CBD constructs (CBDclos, CBDcex, CBDcipA, CBDcenA)
  inserted into pUS250
* Performed colony PCR
* Resolved issues which arose with transfecting cells with pUS250
* Induced cells with cumate and measured fluorescence

##### HP and Modelling

* Discussed what needs to be done and set deadlines for the
  faculty of science stall for University of Sydney’s welcome week
* Docking of sfGFP and nanobody H attempted in OpenMM, with little
  success

1/8/2022 - 7/8/2022

##### Nanobody surface display

* Cloned and transformed the 5 nanobodies into TOP10
  *E. coli*
* Performed PCR on both the nanobodies and neae construct g-blocks
  to use product in further experiments

##### fuGFP-CBD

* Purified fuGFP-CBD samples and ran SDS-Page of 6 fuGFP-CBD
  samples including flow through and washes
* Performed another 2 SDS-PAGEs and fluorescence plate readings to
  test CBD construct binding to filter paper discs and cotton
  balls

##### HP and Modelling

* Ran
  [faculty of science stall](communication#sciweekedu)
  for two days
* Attended information session for science week stall at the
  Australian museum

8/8/2022 - 14/8/2022

##### Nanobody surface display

* Performed colony PCR on nanobody cloning/transformation products

##### fuGFP-CBD

* Ran SDS-PAGE and fluorescence plate readings to compare
  different induction concentrations using cumate
* Repeated assay using cotton and compared induced and uninduced
  samples of the 4 fuGFP-CBD constructs
* Determined optimal induction time by running a time course assay

##### HP and Modelling

* After weeks of trying, OpenMM abandoned as a tool to model the
  docking of GFPs and nanobodies, owing to a lack of success and
  an overabundance of errors

15/8/2022 - 21/8/2022

##### Nanobody surface display

* Tested nanobody binding to sfGFP and fuGFP

##### fuGFP-CBD

* Ran SDS-PAGE of both induced and uninduced of 4 fuGFP-CBDs
  tested before, including the insoluble fraction for
  fuGFP-CBDcipA (brightest sample)
* fuGFP-CBD samples need linker as most of the construct was in
  the insoluble fraction

##### HP and Modelling

* Ran [science week stall](communication#sciweekedu) at
  the Australian museum
* LightDock, an alternative molecular dynamics simulation tool,
  explored as a successor to OpenMM

22/8/2022 - 28/8/2022

##### Nanobody surface display

* First half of DNA shuffling protocol (digest, and reassembly) on
  five nanobody gBlocks

##### fuGFP-CBD

* Amplified and ran SDS-PAGE of fuGFP-linker-CBDs (newly ordered
  constructs)
* Performed golden gate cloning and colony PCR of fuGFP-linker-CBD
  constructs

##### HP and Modelling

* Meeting with Groningen iGEM team

29/8/2022 - 4/9/2022

##### Nanobody surface display

* Tested nanobody and neae surface display binding with purified
  GFP
* Amplification of shuffled products via PCR

##### fuGFP-CBD

* Ran SDS-PAGEs and fluorescence plates of fuGFP-linker-CBDs of
  induced and uninduced samples including the lysate, whole cell,
  and insoluble fraction
* Repeated above assay comparing filter paper discs, cotton, and
  microcrystalline cellulose

##### HP and Modelling

* Meeting with Groningen iGEM team

5/9/2022 - 11/9/2022

##### Nanobody surface display

* Repeat of PCR amplification and electrophoresis gel of shuffled
  products

##### fuGFP-CBD

* Characterised fuGFP-linker-fuGFP binding to cellulose
* Cloned and ran colony PCR of fuGFP-linkerCBDs into pET28ct and
  transformed BL-21(DE3) cells as fluorescence was weak using
  pUS250
* Ran SDS-PAGE of fuGFP-linker-CBDcipA samples (brightest
  fluorescence) binding to microcrystalline cellulose
* Used microcrystalline cellulose from now on

##### HP and Modelling

* Meeting with Groningen iGEM team
* The first successful docking of sfGFP and nanobody H completed
  in LightDock

12/9/2022 - 18/9/2022

##### Nanobody surface display

* Transformed shuffled products and plated on X-GAL and IPTG
* Cloned shuffled products into pUS250-Neae plasmid

##### fuGFP-CBD

* Ran fluorescence plate of 4 fuGFP-linker-CBD constructs using
  different types of buffers to determine the optimal condition
* Use NT from now on

##### HP and Modelling

* Docking of all combinations of GFPs and nanobodies commences,
  with significant runtimes

19/9/2022 - 25/9/2022

##### Nanobody surface display

* Screening of shuffled library lysate by washing with PBS and
  running a fluorescence plate
* Performed colony PCR of colonies with the shuffled products
* Tested GFP binding with the shuffled products

##### fuGFP-CBD

* Ran fluorescence assay, comparing fuGFP-CBDs and
  fuGFP-linker-CBDs
* Also tested different elution conditions using
  fuGFP-linker-CBDcipA
* Further testing using glucose, maltose, and glycerol based on
  above assay
* Used glucose as the eluent from now on

##### HP and Modelling

* Meet up with Washington iGEM team and came up with the idea to
  do a podcast

26/9/2022 - 2/10/2022

##### Nanobody surface display

* Creation of fuGFP variants (fuGFPy, fuGFPb, and fuBFP) via PCR
* Cloned variant plasmids into *E. coli*
* Ran fluorescence plate to characterise the above variants

##### fuGFP-CBD

* Ran SDS-PAGE and fluorescence plate of fuGFP-linker-CBDcipA
  using NT as the buffer and eluting 4 times using glucose
* Sent SDS-PAGE of eluted bands for mass spectrometry
* Ran SDS-PAGE and fluorescence readings of fuGFP-CBD variants
  (fuGFPb, BFP, fuGFPy) lysate and elutions

##### HP and Modelling

* NSW iGEM team meetup on zoom
* Modelling completed, and nanobody-GFP complexes are visualised
  in PyMOL

3/10/2022 - Wiki Freeze!

##### Nanobody surface display

* Nanobody-GFP binding assay (see
  [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D))
* Sequenced original nanobodies, different fuGFP variants, and
  shuffled nanobodies

##### fuGFP-CBD

* Repeated SDS-PAGE and fluorescence readings of fuGFP-CBD
  variants with one sample changed as it did not have an elution
  band on the gel

##### HP and Modelling

* Recorded podcast with
  [Washington iGEM team](human-practices#washpodcast)
* Meeting with
  [Dr Clare Skinner](human-practices#clareskinner)
* Meeting with
  [Dr Maureen O’Malley](human-practices#maureenomalley)
* Meeting with Groningen iGEM team

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{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
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### Overview

To perform high-throughput screening of mutant nanobodies produced by DNA
shuffling cost-effectively, our goal was to immobilise the target antigen
(fuGFP) onto cellulose. To achieve this goal, we needed to design fusion
proteins that link fuGFP to one of several cellulose-binding domains (CBDs).
Our first attempt at the engineering cycle with the construction of our CBD
fusion parts was resoundingly unsuccessful. This was where we implemented
the iGEM certified engineering cycle, designing an improved part, building
it into our expression vector, testing their binding capacity and
fluorescence and learning from the results by comparison of the two parts
functions.

Our initial research found that the Imperial iGEM Team (2014) created fusion
proteins of sfGFP with CBDs clos, cex, cipA and cenA. Therefore, we decided
to base the CBD segments of our fusion proteins on those four CBDs and
obtained the nucleotide sequences from the iGEM parts registry. We designed
our initial fuGFP-CBD fusions and expressed them in our in-house expression
vector pUS250 *E. coli*. Successful fuGFP-CBD fusion proteins should
be most obviously distinguished by fluorescence under UV.

### First Engineering Cycle

![Iteractive DBTL cycle: design, build, test, learn.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-dbtl.png)

**D:** In our first iteration of fuGFP-CBD fusion proteins,
we placed the sequence for fuGFP upstream of each of the CBDs clos ([BBa\_K4488007](http://parts.igem.org/Part%3ABBa_K4488007)), cex ([BBa\_K4488008](http://parts.igem.org/Part%3ABBa_K4488008)), cipA ([BBa\_K4488009](http://parts.igem.org/Part%3ABBa_K4488009)) and cenA ([BBa\_K4488010](http://parts.igem.org/Part%3ABBa_K4488010)) and separated by a 2 amino acid segment. Furthermore, we added
recognition sites for BsaI and BamHI at the start of our fusion protein
sequences, and XhoI and BsaI sites at the end of the sequence to allow
for cloning into pUS250v3 and pET28c(+) (Figure 1).

![A screenshot from snapgene of the first design of fuGFP-CBD fusion protein sequences. fuGFP is placed upstream of CBDs (clos, cipA, cenA, and cex). Restriction sites were added for BamHI, XhoI, and BsaI.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-snapgene-fugfpcbd.png)

**Figure 1.** First design of fuGFP-CBD fusion protein
sequences. fuGFP is placed upstream of CBDs (clos, cipA, cenA, and
cex). Restriction sites were added for BamHI, XhoI, and BsaI.

**B:** To build our proteins, we ordered synthetic DNA for
each of our 4 fuGFP-CBDs, cloned them into pUS250v3, and transformed
them into TOP10 *E. coli*. We expect that if the fusion proteins
are successfully expressed this would be most easily discerned from the
fluorescence displayed by bacteria.

**T:** Our initial testing included measuring whole cell
fluorescence of transformed *E. coli*. We found that only induced
cells expressing fuGFP-CBDcipA were significantly more fluorescent than
uninduced cells (Figure 2). Very minor or insignificant differences in
fluorescence were observed for cells expressing the other 3 fuGFP-CBD
designs.

![Scratch plate of initial transformants of fuGFP-CBD constructs grown on a LB kanamycin 50 μg/mL.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-firstcycleplate.png)

**Figure 2.** Scratch plate of initial transformants of
fuGFP-CBD constructs grown on a LB kanamycin 50 μg/mL. Constructs are
induced in the presence of cumate 100 μmol/mL.

**L:** We hypothesised that the unexpected results were
caused by misfolding of fuGFP, as we adapted the fusion proteins designs
from a previous iGEM team, who used sfGFP instead of fuGFP. sfGFP folds
reliably in native conditions (Pédelacq *et al.* 2006), whereas
fuGFP has not been reported to do so. We believe that the close
proximity between the CBD and fuGFP domains may interfere with protein
folding of the fuGFP beta-barrel, resulting in quenching of the
fluorophore and the poor fluorescence observed from most constructs.

### Second Engineering Cycle

![Iteractive DBTL cycle: design, build, test, learn.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-dbtl-purple.png)

**D:** Existing literature suggests flexible glycine-serine
linkers can be used to improve the stability and folding of fusion
proteins (Chen *et al.* 2013). Therefore, to improve the
functionality of our fusion proteins, we redesigned the region between
the two functional domains, adding a flexible (GGGGS)3 linker
between the GFP C terminus and the CBD N terminus in the second
interaction of our fusion protein (Figure 3). We expected that cells
expressing the new fuGFP-linker-CBD construct would be more fluorescent.

![A screenshot from snapgene of the second design of fuGFP-CBD fusion protein sequences. A flexible (GGGGS)3 linker was placed between fuGFP and CBD domains.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-snapgene-fugfplinkercbd.png)

**Figure 3.** Second design of fuGFP-CBD fusion protein
sequences. A flexible (GGGGS)3 linker was placed between
fuGFP and CBD domains.

**B:** To build our fusion proteins, we ordered synthetic
DNA for our new designs, cloned them into pUS250v3, and transformed
TOP10 *E. coli* for testing. The new parts were named
*fuGFP-linker-CBDclos* ([BBa\_K4488011](http://parts.igem.org/Part%3ABBa_K4488011)), *fuGFP-linker-CBDcex* ([BBa\_K4488012](http://parts.igem.org/Part%3ABBa_K4488012)), *fuGFP-linker-CBDcipA* ([BBa\_K4488013](http://parts.igem.org/Part%3ABBa_K4488013)) and *fuGFP-linker-CBDcenA* ([BBa\_K4488014](http://parts.igem.org/Part%3ABBa_K4488014))

**T:** When testing the fluorescence of both uninduced and
induced *E. coli* expressing the fusion proteins we found that
all *E. coli* expressing the improved
*fuGFP-linker-CBD* had higher average fluorescence compared to
*E. coli* expressing the respective original fuGFP-CBDs without a
linker (Figure 4). Furthermore, significant increases in fluorescence
were observed between induced *E. coli* expressing
*fuGFP-linker-CBD* compared to uninduced *E. coli*. We
also found that cells expressing *fuGFP-linker-CBDcipA* were the
most fluorescent overall.

![A bar chart showing relative fluorescence measurements for fuGFP-CBD without a linker, and a graph for improved fuGFP-linker-CBD, side-by-side.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-fluorescence.png)

**Figure 4.** Relative fluorescence measurements for
fuGFP-CBD without a linker (top) and improved fuGFP-linker-CBD (bottom).
200 μL of *E. coli* culture for each construct was grown in a
96-well plate in LB with kanamycin (50 μg/mL) and either induced with
cumate (100 μM) or not induced for 11 hours. The fluorescence at the end
of 11 hours was divided by OD600 to obtain values for
comparison.

**L:** Cumulatively, we learned from these results that we
successfully improved our original fusion protein designs through
re-engineering with the addition of a flexible linker and recorded their
improved functionality through improved fluorescence (Figure 5). Further
experiments show that these improved fusion proteins also bind strongly
to cellulose and are eluted with glucose and we have described these
findings on our main
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

![A screenshot from snapgene of the second design of fuGFP-CBD fusion protein sequences. A flexible (GGGGS)3 linker was placed between fuGFP and CBD domains.](https://static.igem.wiki/teams/4488/wiki/photos/engineering-patchplate.png)

**Figure 5.** LB-kanamycin patch plates of fuGFP-CBDs
(left) and fuGFP-linker-CBDs with improved fluorescence(right). 25 μL
of 100 mM cumate was added to each plate to induce protein expression.

---

**References**

Chen, X., Zaro, J.L. and Shen, W.-C. 2013, ‘Fusion protein linkers:
Property, design and functionality’,
*Advanced Drug Delivery Reviews*, *65*(10), 1357-1369.

Imperial iGEM Team (2014).,
*Team:Imperial/Functionalisation - 2014.igem.org*, 2014.igem.org.

Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T.C., and Waldo, G.S.
2006. Engineering and characterization of a superfolder green fluorescent
protein. *Nature Biotechnology*, *24*(1), 79-88.

{% endblock %}

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![Flowchart of in-vitro nanobody generating system workflow. It documents every overall procedure an experiment we have used to produce our results](https://static.igem.wiki/teams/4488/wiki/photos/in-vitro-flowchart.png)

* [Overview](#overview)

* [Nanobody Surface Display](#nanobodysurfacedisplay)

* [fuGFP-CBD Fusion](#fuGFPCBDfusion)

* [Creation of fuGFP Variants](#fuGFPvariants)

### Overview

Our project was divided into two wet-lab streams: Surface Display of
Nanobodies and Construction of fuGFP-Cellulose Binding Domains
fusion protein. These streams would come together to allow for the
screening of novel nanobodies against GFPs generated through DNA
shuffling.

---

### Nanobody Surface Display

The surface display side of our project focused on the expression and
evolution of anti-sfGFP nanobodies to anti-fuGFP nanobodies through
DNA shuffling.

Experiments on the surface display side of our project had the
following main aims:

1. Firstly, we needed to clone, transform and establish the function of
   Neae-intimin surface display construct in TOP10 *E. coli*.
2. Secondly, we aimed to express anti-GFP nanobodies on our surface
   display construct, and have them bind sfGFP.
3. Thirdly, we aimed to use DNA shuffling to perform randomised
   evolution to generate a library of shuffled nanobodies.
4. Fourthly, we aimed to screen these shuffled nanobodies for clones
   that bind fuGFP.
5. Fifthly, given the time, we aimed to use modelling data and repeat
   and our shuffling again with new input sequences, until we reach a
   nanobody that strongly binds fuGFP but not sfGFP.

Experimentally, we were able to carry out all our aims through to the
fourth step, and partway through the fifth step. These experiments are
discussed in detail below:

**Parts Design**

The design for our final surface display construct followed Salema and
Fernandez’s 2017 paper which detailed a process for displaying
nanobodies on the surface of *E. coli* using Neae-intimin, a
membrane protein found naturally in *E. coli*. We selected five
anti-sfGFP nanobodies from the literature that we wanted to use in
combination with Neae-intimin. Because of this, we wanted to create a
system where we could first clone in the Neae-intimin, and then in a
second phase clone in our nanobodies.

Surface display part

To design the Neae-intimin part, we adapted the Neae sequence from
literature on Neae-Intimin for surface display (Salema & Fernandez
2017; Salema *et al.* 2013) and inserted a lacZ-alpha operon in
place of the nanobody. We also inserted a flexible linker and a His
tag in between the end of the Neae sequence and the lacZ-alpha
sequence. On either side of the lacZ-alpha sequence, we put in Esp3I
restriction sites that have matching overhangs with each of our
nanobodies. This means that once the Neae part is inserted into our
plasmid, we would be able to insert any one of our nanobody sequences
using Golden Gate cloning. We removed any restriction sites from these
sequences that would interfere with the BioBrick standard. Finally on
either side of the Neae-intimin sequence, outside of the Esp3I sites,
we put BsaI Golden Gate sites with overhangs that match the pUS250
plasmid backbone, as well as our standard adaptor sequences on the
outside which allow us to use standard primers to amplify the ordered
dsDNA.

![An illustration of Neae sequence and the lacZ-alpha sequence with a linker in between and BsaI Golden Gate sites at two ends.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure1.png)

Nanobody part

Through a literature search, we identified five different anti-GFP
nanobodies to display on our *E. coli*. The five anti-GFP
nanobodies were: AIF28002 (NB2), AIF28003 (NB3), AIF28006 (NB6),
AIF28007 (NB7) - denoted by their NCBI codes - and 3OGOH (NBH) -
denoted by its PDB code. The first four are all from the same paper
(Twair *et al.* 2014) and the latter, 3OGOH, is from another
(Kubala *et al.* 2010). To convert these sequences into parts,
we removed restriction sites from the sequences, optimising any
changes for expression in *E. coli*, and added Esp3I sites with
overhangs compatible with our Neae part on either end. Finally, we
added our standard adaptor sequences to either end.

![An illustration of Nanobody sequence with Esp3I sites at two ends.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure2.png)

Double blue-white screening

In the design of our Neae and nanobody parts, we utilised two layers
of blue-white screening. The first layer of blue-white screening was
from the amilCP gene present in our plasmid backbone, which produces a
dark blue, almost purple color. The second layer of blue-white
screening was from the lacZ-alpha operon present in our Neae part,
which in combination with the chemical X-Gal, produces an aqua blue
pigment. These two different kinds of blue white screening allowed us
to screen colonies at both stages of our cloning strategy.

**Neae Cloning and Transformation**

The first step in the lab-work side of creating this surface display
construct was cloning our Neae-lacZalpha part into our plasmid
backbone, pUS250. This part contains the Neae-intimin protein complex
which will display other proteins on the surface of the cell. In place
of the displayed protein, we have inserted a lacZ-alpha gene flanked
by two Golden Gate sites, allowing us to clone in our nanobodies and
our surface display proteins in two different stages.

In order to clone our Neae-lacZalpha surface display complex into our
plasmid backbone pUS250, we used the Golden Gate enzyme BsaI. We set
up a Golden Gate reaction following the basic protocol listed on the
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)
of our wiki.

Once we had created the pUS250-Neae plasmid with Golden Gate, we used
heatshock transformation to transform the new plasmid into TOP10
*E. coli*. We then spread the ligation mixture onto LB agar
plates that contained 50 μM kanamycin, which was the selective marker
for pUS250.

![An illustration of cloning and transformation of Neae G-block into pUS250.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure3.png)

**Screening of Transfomants**

Our plasmid backbone, pUS250, contains an amilcp region between its
two multi-cloning sites. This gene makes *E. coli* create a
dark blue, almost purple pigment. We know that any successful
restriction cloning would have cut out that amilcp region, and
replaced it with the insert. This means that any colonies on our
transformation spread plates from the previous step that are amilCP
blue do not have our Neae insert. Any colonies that are white, either
have pUS250 Neae, or have no plasmid at all. We can be fairly certain
that the former is the case for any white colonies, because if there
was no plasmid present, there would be no conferred antibiotic
resistance and the colony would not have grown. At this stage we had
mistakenly plated onto X-Gal plates without IPTG. So while we had
expected successful colonies to be lacZalpha blue, they were in fact
white instead, due to the lack of IPTG. Some colonies with leaky
expression turned out lacZalpha blue, but junction PCR (as below)
confirmed lacZalpha blue and white colonies were identical. Once we
realised our mistake, we re-plated our transformed cells on plates
containing both X-Gal and IPTG. We tried a few different combinations
of X-Gal and IPTG on the agar plates to figure out what combination of
concentrations worked the best for our plasmid. We found that
spreading 40 μL of 20 mg/mL X-Gal followed by 8 μL of 0.5 M IPTG
produced the best blue-white screening results.

To further confirm which colonies contained our plasmid with the Neae
insert, we performed a junction colony PCR on a few of the white
colonies. To do this we used primers NVC15b and CFM23. Once we
confirmed which colonies carried our successfully cloned plasmid, we
made 5 mL broths of two of those colonies and purified the plasmid via
the plasmid miniprep protocol.

.
![An illustration of screening of transformed colonies.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure4.png)

**Nanobody Cloning**

The second stage of cloning for this construct relies on the Esp3I
Golden Gate sites that we included in our Neae-lacZalpha part. These
sites allow us to clone in any one of our nanobody parts, and be able
to use X-Gal blue-white screening to see which colonies contain
successful clones.

We set up 5 different Golden Gate cloning reactions using Esp3I to
clone in each of our 5 nanobodies (note: when setting up Golden Gate
cloning with Esp3I, it is important to add ATP, as per our basic
protocol). This yielded 5 different versions of the same plasmid, one
for each of the five anti-GFP nanobodies (NBH, NB2, NB3, NB6, and
NB7).

After creating these 5 different plasmids through Golden Gate cloning,
they were each put into TOP10 *E. coli* through heat shock
transformation. These transformations were plated onto IPTG X-Gal
plates according to the concentrations we had previously optimised (40
μL of 20 mg/mL X-Gal, 40 μL of 0.1 M IPTG) so that colonies where
cloning was unsuccessful would appear lacZalpha blue.

Once we had identified a few potentially successful colonies for each
of the five nanobodies via the X-Gal blue-white screening, we
performed a junction colony PCR using the primers iG22-1 and iG22-4 to
confirm with more certainty which colonies contained plasmids with our
inserts. Once these colonies were identified, their plasmids were
purified via miniprep.

![An illustration of cloning of Nanobody G-blocks into pUS250-Neae.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure5.png)

**nb-gfp binding assay**

In order to assess the success of our *E. coli* in expressing
our construct, we devised a Nanobody-GFP binding assay (see
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) for a
granular description). 5 mL LB broths were inoculated with each
nanobody containing-clone, and grown up overnight. ODs were measured
and recorded for standardisation. 1 mL of cells were then pelleted and
resuspended in equimolar amount of sfGFP, fuGFP and eGFPs. This was
calculated assuming 7000 molecules of Neae expressing per cell, a
number obtained from the literature (Salema *et al.* 2017).
Cells were then washed in PBS by pelleting and resuspending. 50 μL of
cells resuspended in PBS after two rounds of washing were then loaded
onto a plate for plate reader fluorescence analysis. Measurements were
taken at 400 nm for the fuGFP (which absorbs blue wavelengths of
light) and at 470 nm for the sfGFP and eGFP. Results showed that four
out of the five nanobodies appeared to be binding sfGFP, as intended,
and none appeared to bind fuGFP. Only Nanobody H was binding eGFP.

As nanobody H was binding most strongly to sfGFP, we proceeded with
another round of the assay for this nanobody in particular to validate
it as a positive control for later testing of shuffled nanobodies.
pUS250-Neae was also verified as a valid negative control at the same
time. This was done with five replicates. Induced versus uninduced
broths were added as another negative control.

As this assay was an invention of our team, it went through various
iterations and refining each time we performed it, in order to
strengthen the assay. The first round was done with no replicates, but
each subsequent assay was done at least in duplicate, if not in
triplicate or with five replicates. Induced versus uninduced broths
were added as another negative control, and later, no GFP added
negative controls were performed as well. By the time it came to test
the binding of our shuffled nanobodies (as detailed in a later section
of the experiments), we were fairly confident in the assay’s efficacy
and ability to qualitatively determine whether a nanobody was binding
strongly to a GFP or not.

**DNA Shuffling**

DNA shuffling (a.k.a. sexual PCR) is a method in which fragments of
similar pieces of DNA are mixed together, cut in many places, and then
reassembled at random. The goal of this process is to create many new
variations on a given gene or fragment via a kind of artificial
meiosis. Our project aimed to use DNA shuffling to create many random
variants of anti-sfGFP nanobodies, with the eventual goal of finding a
variant that would bind to fuGFP instead of sfGFP. This method was
chosen because DNA shuffling was the method used to originally create
fuGFP from sfGFP (Small Things Considered 2019).

The protocol we used to perform DNA shuffling was published in 2014 by
Meyer *et al.*. This protocol was implemented with very few
changes, but for clarity's sake you can find our entire detailed
process written out in our protocols page.

The first step in DNA shuffling is to acquire a few different similar
variants on the same gene. We did this in our
[parts design phase](#design), where we found 5 different
sequences for anti-sfGFP nanobodies, and ordered them as gBlocks. When
designing these parts, we also created two sets of primers to be used
in shuffling. One was the outer set of primers (CFM 129 and CFM130
which bind in the standard adaptor region flanking our nanobody
parts), which is used in the initial amplification of fragments
pre-shuffling, and the other was the inner set of primers, used to
amplify the post-shuffling fragments. The two sets of primers need to
be distinct because the shuffling process can degrade the ends of the
DNA fragments, meaning that the annealing sites of the outer primers
may not still be present after shuffling.

Using the outer set of primers, we PCR amplified each of our five
anti-sfGFP nanobodies from our ordered gBlocks. These nanobodies were
all combined in equal parts to create the DNA mixture that was put
through the shuffling protocol.

The detailed, and reproducible, version of the shuffling protocol can
be found here, but for those of you that want a more general overview,
here a quick summary: First the five nanobody sequences (after being
mixed in a tube and amplified via PCR) are cut up into small pieces by
a nonspecific DNAse. Then these pieces are ligated back together. In
this process, pieces from one nanobody will be ligated to pieces from
another. This process is kind of like the crossing over you would find
in the process of meiosis, resulting in pieces of DNA that are a
mixture of the input fragments.

Once the shuffling protocol was completed, we amplified the final mix
using the inner set of primers using PCR (iG22-3, iG22-4) Because the
annealing sites of these primers include the Esp3I type IIS
restriction site, this PCR yields a large collection of different
nanobodies which still have the relevant Golden Gate cloning sites and
overhangs.

Finally, this PCR product was purified via spin column purification to
prepare it for cloning in the next phase of the project.

![An illustration of shuffling of ordered nanobodies.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure6.png)

**Shuffled NB Cloning**

In much the same way as the cloning of the original five nanobodies
into pUS250-Neae, the mixture of newly shuffled nanobodies (the
purified PCR product from the last step of
[DNA shuffling](#shuffling)) were inserted via Golden Gate
cloning into pUS250-Neae using Esp3I. Then the resulting plasmids were
then transformed via heat shock transformation into TOP10
*E. coli*. In order to generate a large enough library to
screen for a colony that may bind fuGFP, 16 plates of transformants
were plated, generating a library of approximately ~10k colonies.

![16 plates of transformed colonies of pUS250-Neae-shuffled nanobodies into TOP10 E. coli.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure7.png)

**Above:** 16 plates of transformed colonies of
pUS250-Neae-shuffled nanobodies into TOP10 *E. coli*.

![An illustration of screening of transformed colonies.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure8.png)

Out of these plates, plate number 12 was an IPTG X-Gal control plate
so that we could tell the efficiency of the clongin via blue-white
screening. Unfortunately this batch of X-Gal IPTG plates were not
functional and yielded no blue colonies at all.

Foolishly, we presumed that this meant that our transformation had
just gone exceptionally well, and proceeded with sequencing and our
screening protocol.

To prepare the samples for sequencing, we did a spanning PCR with
primers iG22-1 and CFM4 on either side of the shuffled nanobody insert
region. We then purified this PCR product and sent it for sanger
sequencing with the primer iG22-2, which would amplify the nanobody
region. When the sequencing results came back, we found that all of
the colonies that we sequenced did not contain an insert at all, let
alone a shuffled nanobody.

![An illustration of cloning of shuffled nanobodies into pUS250 backbone.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-nb-figure9.png)

**Shuffled NB Screening**

In order to screen the shuffled nanobodies for binding to fuGFP, a
protocol was developed by our team that harnessed the
fuGFP-linker-cellulose binding domain fusion proteins the GFP-CBD side
of our project had developed. See protocols for granular detail. The
library was recovered and homogenised. Aliquots were then used to
inoculate 50 mL broths of LB Km50, with 100 μM cumate to induce the
expression of the constructs. This was then put through a column with
a cotton wool plug with immobilised fuGFP-CBDs attached. 10 mL of
cells were added to the column at a time. Once all cells had been put
through the column, 10 mL of PBS was added to the column at a time,
the idea being that cells expressing a nanobody that would be able to
bind fuGFP would not be washed off, but those that did not bind would.
Flow through was collected and measured for OD until the OD reached
near enough to zero.

The cotton wool was then removed from the column, and placed in a 50
mL LB broth to grow up a number of cycles. Cells were then recovered
from the broth and plated out onto LB Km50 plates, and incubated
overnight. This resulted in 19 colonies that had made it through the
screening. A pellet of cells was also generated and frozen.

These cells were then prepared for sequencing, using the same methods
as above (primers iG22-1 and CFM4).

Simultaneously, the cells were put through the Nanobody-GFP binding
assay, a few at a time. Only a number had been screened when
sequencing results returned, showing that all 19 colonies were in fact
the original pUS250-Neae plasmid, with no nanobody insert.

While this was disheartening, it was clear that due to the failed
blue-white screening, we had failed to identify that our cloning
efficiency was extremely low, and plasmid background therefore
extremely high. In order to deal with this, the previously frozen
pellet was resuspended and plasmid prepped, before an Esp3I digest was
performed. As we had used golden gate cloning, successfully cloned
plasmids would no longer contain the Esp3I sites, while the
pUS250-Neae backbone retained them. This was then transformed into
TOP10 *E. coli*, resulting in a plate of nine colonies.

A spanning PCR was performed on these nine colonies using the primers
iG22-1 and MVS77 to prepare for sequencing. Spanning PCR results
showed that only colony number 7 had an insert that was the right
length to be a functional nanobody. In order to get some idea of the
diversity of the sequences before sequencing results returned, an MspI
digest was performed on the nine colonies and also the five original
anti-GFP nanobodies, and run out on a gel. From this it was identified
that colony number 7 appeared to have a unique digest pattern
different to any of the original anti-GFP nanobodies, however the
unique band that differentiated it from nanobody three was very faint
and only around 20-50bp in length, so we could not be certain. Colony
seven was named Allocamelus to differentiate it from the original
nanobody number 7.

We proceeded to perform the Nanobody-GFP binding assay on Allocamelus
in order to ascertain whether or not it bound to fuGFP or sfGFP. This
assay was run with nanobody H as a positive control for binding to
sfGFP, and nanobody three as based on the MspI digest gel, there was a
small chance Allocamelus was unshuffled nanobody three. Induced versus
uninduced and no GFP controls were also performed. While this did not
show any signs of Allocamelus binding fuGFP, it also did not bind
sfGFP, while nanobody three does.

Sequencing results were able to confirm that colony 7, A.K.A.
Allocamelus (see
[Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) for
more information) was indeed a successfully shuffled colony.

Why Allocamelus?

![ye olde image of an Allocamelus, a mythical creature with the head of a donkey and a camel's body](https://static.igem.wiki/teams/4488/wiki/photos/why-allocamelus.png)

The allocamelus or ass-camel is a mythical creature with the
head of a donkey or mule and the body of a camel (Topsell 1658).
This creature features in British heraldry (Smith 1928). We
named our one successfully shuffled nanobody after this strange
beast because our nanobodies sequences come originally from
camelids (of which the allocamelus is one) and that they are
shuffled, much like the camel and donkey are shuffled together
to create the allocamelus. Although it probably wasn’t necessary
to give this shuffled nanobody such a fanciful name, we decided
to have a bit of fun with it.

After sequencing our “allocamelus” nanobody, we realised that it
fit better with the name than we ever anticipated! It turns out
that this nanobody was a mix of just two of our original
nanobodies (NB2 and NB3), just like how allocamelus is a mix
between two animals.

Smith, R. F. (1928).
*An Early Map of Surrey. The British Museum Quarterly,
3(1)*, 16. doi:10.2307/4420919

Topsell, E. (1658).
*History of Four-footed Beasts and Serpents*.

---

### fuGFP-CBD Fusion

**Initial Designs**

We started our fusion protein design by searching the parts registry
for existing work on cellulose-binding domains. We found that the 2014
Imperial iGEM team Aqualose had previously experimented with CBDs
fused to sfGFP and chose 4 different CBDs (cenA, cipA, cex, and clos)
to incorporate in our designs. The sequences for our initial
fuGFP-CBDs contain the sequence for fuGFP upstream of a CBD sequences
and connected by a 2 amino acid gap. Furthermore, we inserted
recognition sites for BsaI and BamHI at the start of our fusion
protein sequences, and XhoI and BsaI sites at the end of the sequence
to allow for cloning into pUS250v3 and pET28c(+).

![An illustration of fuGFP and CBD sequences with BsaI sites at two ends.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-cbd-figure1.png)

**Cloning and transformation**

Synthetic DNA of our fuGFP-CBD sequences were obtained and cloned into
pUS250v3 via golden gat cloning with BsaI and transformed into TOP10
*E. coli* using heat-shock. We performed a colony PCR on
transformed cells using primers MVS48 and CFM23 which bind in the
pUS250v3 backbone on opposite ends of the insert sequence. The results
from agarose gel electrophoresis showed most of the products matched
our expected insert sequence of ~1100 bp, indicating a high cloning
and transformation success rate.

![An illustration of cloning of fuGFP-CBDs into pUS250 backbone.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-cbd-figure2.png)

**Initial growth and testing**

After confirming TOP10 *E. coli* were successfully transformed,
we tried small-scale expression of our fusion proteins using 5 mL
cultures and tested whether they were fluorescent and binding
cellulose. We used bead beating to extract cell lysate from
*E. coli* expressing fuGFP-CBDs we added these to filter disk
paper as well as microcrystalline cellulose. We then measured changes
of fluorescence to the cellulose and performed SDS-PAGE to determine
whether proteins were bound to cellulose as well as the amount of
fuGFP-CBDs being expressed by TOP10 *E. coli*. Overall we found
that only fuGFP-CBDcipA showed caused cellulose to gain any
significant fluorescence.

![An illustration of fluorescence assays and SDS-PAGE gels.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-cbd-figure3.png)

**Redesign and addition of a flexible linker**

In an attempt to improve the functionalisation as well as expression
of our fusion proteins, we redesigned the initial fuGFP-CBD sequences
by adding a flexible (GGGGS)3 linker ([BBa\_K4488006](http://parts.igem.org/Part%3ABBa_K4488006)) between fuGFP and each of the separate CBDs. The new
fuGFP-linker-CBDs were cloned and transformed into TOP10 for initial
testing. Colony PCR using primers MVS48 and MVS71 which bind in the
plasmid backbone and fuGFP sequence respectively, again confirmed
successful transformation into TOP10.

![An illustration of re-design of fuGFP-CBD parts with a linker, showing fuGFP and CBD sequences with a linker in between, in new sequence there are BamHI and XhoI sites at two ends.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-cbd-figure4.png)

**Growth and testing in BL21(DE3)**

To improve the overexpression of fuGFP-linker-CBDs, we cloned the
fusion protein sequences into pET28c(+) using BamHI and XhoI
restriction enzymes and transformed BL21(DE3) using heat-shock.
BL21(DE3) is a common *E. coli* strain for recombinant protein
expression because of it has been engineered to produce the highly
efficacious T7 polymerase and lacks proteases (Ratelade
*et al.* 2009). Expression with 100 mL broths of BL21(DE3)
instead of TOP10 *E. coli* massively increased our protein
yield. Furthermore, we chose to focus on fuGFP-linker-CBDcipA since
all our previous experiments suggested this was the most functional in
terms of cellulose-binding and fluorescence. We were able to show that
fuGFP-linker-CBDcipA binds to cellulose through our tests.

We were also interested in determining whether fuGFP-linker-CBDs could
be selectively eluted after being bound to cellulose as this provides
potential applications for using fuGFP-CBDs as a tag for protein
purification. Previous studies have found that CBD fusion proteins can
be purified by binding to cellulose and eluted with solutions low in
ionic strength such as distilled water (Sugimoto
*et al.* 2012). We hypothesised that molecules structurally
similar to cellulose may be able to compete for binding to CBD domains
and cause the fuGFP-CBDs to be eluted from cellulose.

Using our protocol with fuGFP-linker-CBD bound to microcrystalline
cellulose, we screened elution conditions using cellobiose, maltose,
glucose, glycerol, and distilled water. We found that glucose with a
concentration of 1 M (or above) is most effective for eluting
fuGFP-linker-CBDcipA resulting in fractions with high purity. Overall,
our experiments have shown that fuGFP-CBDs bind to cellulose and are
eluted with glucose which may be useful in protein screening or
purification.

---

### Creation of fuGFP Variants

**Creation of new free-use fluorescent proteins**

Under the guidance of our advisor Mark Somerville, we explored the
possibility that the fluorophore of fuGFP could be mutated to generate
other fluorescent proteins with differing Excitation/Emission spectra.
This aligned with feedback we’d received on our project (see
[Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)). These mutations had been characterised and were known in other
GFPs (Tsien 1998).

With assistance from our advisor, we used the degenerate primer MVS70
(with redundancy at three loci of the fluorophore) to amplify plasmid
preps of pUS252-fuGFP and our pET28-fuGFP-linker-CBDs and generate
variant fluorescent proteins.

Primer: AAACGTCTCCCTTGRSCYATGGCGTGCAG

We then performed a PCR cleanup (see
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)). The PCR products were then digested with DpnI to remove any
unamplified plasmid, then with Esp3I restriction enzyme to be
recircularised with Hi-T4 Ligase. The resultant mixture of plasmids
were then transformed into appropriate hosts - pUS252-fuGFPx into
TOP10 *E. coli* and pET28-fuGFP-linker-CBD into BL21
*E. coli*. We varied the amount of ligation mix added to our
transformations - either 2 or 4 µL - as we were not sure what would be
the most efficient. This produced a plates of transformants that could
then be analysed through a plate-reader fluorescence assay and for
binding and eluting to cellulose (see
[Protocols page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)
for relevant protocols).

![An illustration of creation of fuGFP variants after PCR to fuBFP, fuGFPb, fuGFPy, and fuGFP-alanine.](https://static.igem.wiki/teams/4488/wiki/photos/experiment-var-figure1.png)

---

**References**

Kubala, M. H., Kovtun, O., Alexandrov, K., & Collins, B. M. 2010.
'Structural and thermodynamic analysis of the GFP:GFP-nanobody complex',
*Protein Science*, *19*, 2389-401.

Meyer, A. J., Ellefson, J. W., & Ellington, A. D. 2014. Library
generation by gene shuffling.
*Current protocols in molecular biology*, *105*,
Unit–15.12.
<https://doi.org/10.1002/0471142727.mb1512s105>

Ratelade, J, Miot, M-C, Johnson, E, Betton, J-M, Mazodier, P &
Benaroudj, N 2009, ‘Production of Recombinant Proteins in the lon
-Deficient BL21(DE3) Strain of *Escherichia coli* in the Absence
of the DnaK Chaperone’, *Applied and Environmental Microbiology*,
vol. 75, no. 11, pp. 3803–3807.

Salema, V., and Fernández LÁ. 2017. 'Escherichia coli surface display
for the selection of nanobodies', *Microb Biotechnol*,
*10*, 1468-84.

Salema, V., Marín E., Martínez-Arteaga R., Ruano-Gallego D., Fraile S.,
Margolles Y., Teira X., Gutierrez C., Bodelón G., & Fernández LÁ. 2013.
'Selection of single domain antibodies from immune libraries displayed
on the surface of E. coli cells with two β-domains of opposite
topologies',
*PLoS One*, *8*: e75126.

Sugimoto, N, Igarashi, K & Samejima, M 2012, ‘Cellulose affinity
purification of fusion proteins tagged with fungal family 1
cellulose-binding domain’, *Protein Expression and Purification*,
vol. 82, no. 2, pp. 290–296.

*The Story of Free Use GFP (fuGFP).* Small Things Considered.
2019.
<https://schaechter.asmblog.org/schaechter/2019/05/the-story-of-free-use-gfp-fugfp.html>.

Tsien, Roger Y. 1998. 'THE GREEN FLUORESCENT PROTEIN',
*Annual Review of Biochemistry*, vol. 67, pp.509-44.

Twair, A., Al-Okla, S., Zarkawi, M., & Abbady, A. Q. 2014.
Characterization of camel nanobodies specific for superfolder GFP fusion
proteins.
*Molecular biology reports*, *41*(10), 6887–6898.
<https://doi.org/10.1007/s11033-014-3575-x>

{% endblock %}

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

* [Overview](#overview)

* [Statistics](#statistics)

* [Nanobody Surface Display](#nanobodysurfacedisplay)

* [fuGFP-CBD Fusion](#fuGFPCBDfusion)

* [Creation of fuGFP Variants](#fuGFPvariants)

* [Contribution: Basic Part Characterisation](#partcontribution)

### Overview

Our project was divided into two wet-lab streams: Surface Display of
Nanobodies and Construction of fuGFP-Cellulose Binding Domains
fusion protein. These streams would come together to allow for the
screening of novel nanobodies against GFPs generated through DNA
shuffling.

---

### Statistics

Statistics were used for data visualisation in both wet-lab streams.
In cases where multiple replicates were sampled, we constructed
generalised linear models using the “glmmTMB” package (Brooks
*et al.* 2017) in R version 4.2.1 (R Core Team 2022). We used
the “testResiduals” function in the “DHARMa” package (Hartig 2022)
to assign model families and test assumptions, and then performed
Wald Chi-square test using the “car” package (Fox & Weisburg 2019)
to extract p-values from models. Where we observed a significant
result, we used the “emmeans” package (Lenth 2022) to perform
Tukey-Kramer post-hoc contrasts.

---

### Nanobody Surface Display

**Summary**

The surface display side of our project focussed on the expression
and evolution of anti-sfGFP nanobodies to anti-fuGFP nanobodies
through DNA shuffling. Expression was done in the TOP10
*E. coli* using pUS250 as our vector. Two layers of golden
gate cloning were used to clone, and the cymR operon was used to
induce expression. Once expression of pre-existing nanobodies had
succeeded, we successfully created a new variant nanobody
*in vitro*
through DNA shuffling, cloned and transformed and expressed the
variant nanobody as well.

**Achievements:**

* Successfully created recombinant *E. coli* which display
  anti-sfGFP nanobodies on their surface, and proved that the
  nanobodies can still bind sfGFP when displayed in this manner.
* Applied DNA shuffling to create a variant sequence based on our
  anti-sfGFP nanobodies. In doing so we have changed the function of
  the nanobody from binding sfGFP to not binding sfGFP.
* Developed a new protocol for testing the binding of GFP to
  displayed nanobodies.

**Anti-sfGFP Nanobodies**

Construction and transformation of Neae-Intimin anti-sfGFP nanobody
vector into *E. coli*

Our first stage focused on inserting our Neae-Intimin Nanobody
construct into TOP10 *E. coli*. This was done through two
stages of cloning and transformations - firstly the Neae-Intimin, then
the nanobodies (see
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
for more detail). At each stage we harnessed phenotypic blue-white
screening and junction PCRs to confirm successfully cloned colonies.

*Creation of surface display plasmid*

In order to display our collection of anti-sfGFP nanobodies, we
created a plasmid which contained Neae-intimin, a bacterial protein
which can display other proteins (i.e. our nanobodies) on the surface
of *E. coli*. As shown in Figure 1, our Neae part contained a
lacz-alpha gene flanked by Golden Gate cloning sites so that we could
clone in our nanobodies after the fact. This means that this first
stage of cloning was just taking the Neae-intimin gene and inserting
it into our plasmid backbone.

![Plasmid map of backbone showing the cloning strategy.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure1.png)

**Figure 1.** Plasmid map of backbone showing the
cloning strategy. The Neae part will replace the amilCP region in
the plasmid backbone. Primer sites for testing for insertion via
junction PCR are shown.

By replacing the amilCP gene in our plasmid backbone with the
Neae-intimin part via Golden Gate cloning, we were able to use the
blue colour produced by amilCP as a method of screening. Any blue
colonies we saw after transformation had the original backbone, and
any white colonies had the Neae-intimin insert.

![Transformants resulting from insertion of Neae-intimin into our pUS250 plasmid backbone via Golden Gate cloning.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure2.png)

**Figure 2.** Transformants resulting from insertion of
Neae-intimin into our pUS250 plasmid backbone via Golden Gate
cloning. Blue colonies result from the presence of amilCP in the
plasmid backbone, whereas white colonies result from the replacement
of amilCP with our Neae-intimin part.

As can be seen in Figure 2, we were able to use amilCP blue white
screening to identify colonies that were likely to contain our Neae
insert. To confirm this, we used junction PCR on a few of these white
colonies. The gel from this colony PCR can be seen in Figure 3.

![A DNA agorse gel showing results of a junction PCR on 11 colonies resulting from the cloning of Neae into the pUS250 backbone.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure3.png)

**Figure 3.** Gel showing results of a junction PCR on
11 colonies resulting from the cloning of Neae into the pUS250
backbone. These colonies were all selected based on amilCP
blue-white screening. The expected product size of this
amplification was ~350 bp, which is reflected in all samples except
for colony 4.

The gel in Figure 3 shows that almost every colony we sampled
amplified with the expected size of ~350 bp. This means that all of
these colonies except for colony 4 had the Neae insert. From the PCR
results and our blue-white screening with amilCP, we concluded that we
had successfully inserted our Neae part into our plasmid backbone
pUS250, and we were ready to progress to the next step of our project.
We purified the plasmid from colony 2 to use as our pUS250-Neae
plasmid for future stages of the project.

*Insertion of anti-sfGFP nanobodies into pUS250-Neae*

Once we had purified our plasmid from the last step, we were ready to
clone in our anti-GFP nanobodies. To do this we used the Esp3I Golden
Gate sites that are present on either side of the lacZalpha region in
our Neae part. This means that when we cloned in our nanobodies, they
replaced the lacz-alpha. We did five Golden Gate clonings and
transformations, one for each of the five anti-sfGFP nanobodies.

![A diagram showing the strategy for cloning our nanobody parts into pUS250-Neae.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure4.png)

**Figure 4.** Diagram showing the strategy for cloning
our nanobody parts into pUS250-Neae. In successful cloning, the
nanobody part replaces the lacZ-alpha region.

In combination with the chemical X-Gal, the lacZalpha gene creates a
blue colour. This means that we were able to use blue-white screening
again to see which colonies had successfully recombinant plasmids.
When plated on agar containing X-Gal and IPTG, the blue colonies were
our Neae plasmid with no insert, and the white colonies contain the
Neae plasmid with a nanobody insert (Figure 5).

![An agar plate with transformants from the insertion of an anti-GFP nanobody into pUS250-Neae.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure5.png)

**Figure 5.** Agar plate with transformants from the
insertion of an anti-GFP nanobody into pUS250-Neae. The blue
colonies contain pUS250-Neae, while the white colonies contain
successfully recombinant plasmids.

Based on the blue-white screening, we concluded that it was very
likely that all of the white colonies contained our insert of
interest. To further confirm this, we performed a junction PCR on a
few white colonies.

![An electrophoresis gel showing the results of a junction PCR testing for the insertion of nanobody fragments in transformant colonies.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure6a.png)
![An electrophoresis gel showing the results of a junction PCR testing for the insertion of nanobody fragments in transformant colonies.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure6b.png)

**Figure 6.** Electrophoresis gel showing the results
of a junction PCR testing for the insertion of nanobody fragments in
transformant colonies. Colonies were selected by X-Gal blue-white
screening. In all cases except for one of the replicates of nanobody
H, and the two negative controls (original pUS250 and pUS250-Neae)
the band was of the expected size of ~500 bp.

The PCR gel (Figure 6) combined with the blue-white screening were
strong evidence that the colonies we had picked did indeed contain our
anti-sfGFP nanobodies. We had 4 colonies for each nanobody, besides H
where we had three, and 7 where we had two (numbered three and four).
We decided to proceed with colony number three for each Nanobody.

Testing the Function of Neae-intimin Construct Displaying
anti-sfGFP Nanobodies

In order to test the function of our construct we performed a GFP
binding assay (see
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D))
where purified GFPs are added to induced cells, then washed off and
measured for fluorescence. Preliminary testing showed that four out of
the five nanobodies appeared to bind to sfGFP, with H binding markedly
stronger than the other nanobodies. This was validated by our
modelling results (see our
[Modelling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)).

![Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 400 nm.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure7.png)

**Figure 7.** Binding of anti-GFP nanobodies H, 2, 3, 6
and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 400
nm (full settings can be found under
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)).
Bars represent values measured from a single replicate. Measurements
at 400 nm are taken to optimise for fuGFP fluorescence. This graph
shows no difference in binding between fuGFP and Neae negative
control.

![Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 470 nm.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure8.png)

**Figure 8.** Binding of anti-GFP nanobodies H, 2, 3, 6
and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 470
nm (full settings can be found under
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)).
Bars represent values measured from a single replicate. This graph
shows strong binding between sfGFP and nanobodies H, 2, 3, and 7
when compared to Neae negative control. Nanobody 6 does not appear
to bind sfGFP more than the negative control. Nanobody H appears to
bind the most to sfGFP.

![Nanobody-GFP Binding Assay to validate H as a positive control and Neae-Intimin as a negative control for the binding to sfGFP.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure9.png)

**Figure 9.** Nanobody-GFP Binding Assay to validate H
as a positive control and Neae-Intimin as a negative control for the
binding to sfGFP. Measurements were taken for a concentrated and
equimolar amount of GFP added to the nanobodies. Bars represent
means ± SE fluorescence at 470 nm, n = 5 for each bar. Results of a
negative binomial family GLM showed a significant interaction
between nanobody ID and sfGFP concentration (χ23 = 10.9,
p = 0.013). Results of post-hoc contrasts are shown in the plot,
with any bars not sharing a letter being significantly different.

The next step was to validate the Neae-intimin construct as a negative
control and nanobody H as the positive control for testing of shuffled
nanobodies later on. We repeated the Nanobody-GFP binding assay with 5
replicates for each data point for just sfGFP. Induced and uninduced
cells expressing just Neae with no nanobody and Neae-NBH were tested.
Two different concentrations were measured to refine the assay. Figure
9 shows this. Significances were calculated from a tukeyHSD post-hoc
contrast. No significant difference was found between uninduced
versions of Neae and H. A significant difference was found between the
induced nanobody H binding and all other data points. This validated
the use of H as a positive control.

**DNA Shuffling of anti-sfGFP Nanobodies**

Based on the above results, we were confident that our surface display
method of testing nanobody binding was successful. The next step of
this project was to create variations on these nanobodies using DNA
shuffling, and test the difference in their function.

DNA Shuffling

We shuffled together all of the five anti-sfGFP nanobody sequences
that we obtained from the literature. The first stage of this was
mixing the five sequences together in one reaction, and cutting them
with DNAse I. To confirm that this digest was successful, we ran a gel
comparing the product of our digest with the undigested nanobody
sequence.

![Electrophoresis gel confirming the function of our DNAse I digest, which is the first stage of DNA shuffling.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure10.png)

**Figure 10.** Electrophoresis gel confirming the
function of our DNAse I digest, which is the first stage of DNA
shuffling.

The comparison of the undigested fragment mixture and the digested in
Figure 10 clearly shows that the DNAse I digest was successful. Based
on this, we proceed to the next phase of DNA shuffling, which is the
reassembly of the pieces into new fragments. Once we had performed
this reassembly, the new reassembled fragments were PCR amplified.

![Electrophoresis gel showing the DNA shuffling products amplified by PCR. Amplified band was of the expected length of 352 bp.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure11.png)

**Figure 11.** Electrophoresis gel showing the DNA
shuffling products amplified by PCR. Amplified band was of the
expected length of 352 bp.

The gel shown in Figure 11 indicates that there was reassembly of the
digested fragments. This indicates that some complete fragments, like
digested and reassembled nanobodies, were able to be amplified. This
supports the idea that the shuffling steps did proceed successfully,
as the gel results correspond to what we would expect.

We then cloned the shuffled products into pUS250-Neae backbone and
then transformed into TOP10 *E. coli*. From the resulting
control plate on X-Gal IPTG, all colonies appeared white, indicating
efficiency of transformation and successful transformants.

Unfortunately, sequencing results showed a different story. All 11
sequences came back as the lacZ-alpha operon. This indicates that the
blue-white screening failed in this case (the plates were not at the
right concentration of X-Gal IPTG).

A second batch that made it through our screening for nanobodies
binding fuGFP (see screening
[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)) were
sent off for Sanger sequencing. The sequences showed that all colonies
contained the lacZ-alpha operon, meaning that the screening did not
adequately isolate only fuGFP binding nanobodies.

![Results of spanning PCR of 9 colonies containing putatively shuffled nanobodies, as well as colonies containing our 9 original nanobodies pUS250-Neae was used as a negative control.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure12a.png)
![Results of spanning PCR of 9 colonies containing putatively shuffled nanobodies, as well as colonies containing our 9 original nanobodies pUS250-Neae was used as a negative control.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure12b.png)

**Figure 12.** Results of spanning PCR of 9 colonies
containing putatively shuffled nanobodies, as well as colonies
containing our 9 original nanobodies pUS250-Neae was used as a
negative control.

After an Esp3I digest to remove background pUS250-Neae, nine colonies
were isolated. A spanning PCR showed one colony (colony #7) with the
correct length for a shuffled nanobody, named ‘Allocamelus’ by our
team (Figure 12).

![Electrophoresis gel showing the result of a MspI digest of 9 transformant colonies containing putatively shuffled nanobodies, as well as colonies containing the 5 original nanobodies. A colony containing the Neae-lacZ plasmid with no insert was used as a negative control.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure13.png)

**Figure 13.** Electrophoresis gel showing the result
of a MspI digest of 9 transformant colonies containing putatively
shuffled nanobodies, as well as colonies containing the 5 original
nanobodies. A colony containing the Neae-lacZ plasmid with no insert
was used as a negative control.

All nine colonies and the original 5 anti-sfGFP nanobodies were put
through a digest with MspI, and ran out on a gel (Figure 13). The gel
showed a unique pattern of bands for Allocamelus when compared to the
five anti-sfGFP nanobodies. This was a good indication that
Allocamelus may be shuffled.

Why Allocamelus?

![ye olde image of an Allocamelus, a mythical creature with the head of a donkey and a camel's body](https://static.igem.wiki/teams/4488/wiki/photos/why-allocamelus.png)

The allocamelus or ass-camel is a mythical creature with the
head of a donkey or mule and the body of a camel (Topsell,
1658). This creature features in British heraldry (Smith,
1928). We named our one successfully shuffled nanobody after
this strange beast because our nanobodies sequences come
originally from camelids (of which the allocamelus is one) and
that they are shuffled, much like the camel and donkey are
shuffled together to create the allocamelus. Although it
probably wasn’t necessary to give this shuffled nanobody such
a fanciful name, we decided to have a bit of fun with it.

After sequencing our “allocamelus” nanobody, we realised that
it fit better with the name than we ever anticipated! It turns
out that this nanobody was a mix of just two of our original
nanobodies (NB2 and NB3), just like how allocamelus is a mix
between two animals.

Smith, R. F., 1928. An Early Map of Surrey.
*The British Museum Quarterly*, *3*(1), 16.
doi:10.2307/4420919

Topsell, E., 1658.
*History of Four-footed Beasts and Serpents*.

![Diagram showing the genetic makeup and origins of the shuffled nanobody “Allocamelus.”](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure14.png)

**Figure 14.** Diagram showing the genetic makeup and
origins of the shuffled nanobody “Allocamelus.”

Sequencing results for Allocamelus confirmed that it was indeed
shuffled. From base pairs one to 259 Allocamelus matched Nanobody 3,
and from 292 bp to 393 bp Allocamelus matched the sequence for
Nanobody 2 (with the juncture having occurred somewhere in between in
a 33 bp conserved region) providing definitive evidence that the
shuffling did succeed (Figure 14).

![Binding assay for Allocamelus (Nanobody-AC), the shuffled nanobody, measurements taken at 400 nm.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure14a.png)
![Binding assay for Allocamelus (Nanobody-AC), the shuffled nanobody, measurements taken at 470 nm.](https://static.igem.wiki/teams/4488/wiki/photos/results-nb-figure14b.png)

**Figure 15.** Binding assay for our shuffled nanobody
Allocamelus (Nanobody-AC), measured at both 400 nm and 470 nm.
Nanobody H was included as a positive control for binding to sfGFP.
Neae-intimin was included as a negative control for binding to both
sfGFP and fuGFP. No GFP was added to one set of replicates as
another negative control. Bars represent means ± SE, n = 3 for each
bar. Results of gaussian family GLMs showed a significant difference
between nanobodies for 400 nm (χ23 = 27.0, p < 0.001),
and a significant three-way interaction between GFP, nanobody and
induced treatment for 470 nm (χ26 = 508.2, p < 0.001).
Key significant post-hoc contrasts are outlined below in Table 1.

**Table 1.** Summary of key significant contrasts
observed from post-hoc Tukey-Kramer test of 470 nm nanobody binding
test GLM.

| Comparison | p-value |
| --- | --- |
| Allocamelus vs NB3 | 0.0001 |
| Allocamelus vs NBH | < 0.0001 |
| Allocamelus vs Neae | 0.1833 |
| NB3 vs NBH | < 0.0001 |
| NB3 vs Neae | 0.0387 |
| NBH vs Neae | < 0.0001 |

In order to test the function of the Allocamelus nanobody
(nanobodyAC), we performed another GFP binding assay with replicates
(n = 3). The results from this assay show that nanobodyAC does not
bind to sfGFP or fuGFP. Statistical analysis through a tukeyHSD
post-hoc contrast in R showed significant difference in the binding to
sfGFP between Allocamelus and NBH, our positive control, and also
Allocamelus and NB3, the nanobody it shares most of its sequence with,
but not significant difference in binding to sfGFP when compared to
Neaa-intimin, our negative control. As the original nanobodies that
allocamelus is made up of do bind sfGFP, this is a major conclusion in
that we have succeeded in changing the function of a nanobody
utilising DNA shuffling.

**Future Directions**

Given another month or two, we would make more use of the modelling
data we have been given late on in this project. The next step would
be to use modelling to design nanobodies that may be closer to strong
binding to fuGFP, order these as gBlocks and go through the shuffling
process again. This would hopefully generate a diversity of shuffled
nanobodies, that, with fixed blue-white screening, could be put
through the column and tested for binding to fuGFP. This would
increase the likelihood of obtaining a nanobody that does bind fuGFP.

---

### fuGFP-CBD Fusion

**Summary**

We developed fusion proteins of fuGFP bound to cellulose-binding
domains (CBD) which immobilise fuGFP onto a cellulose matrix and
enable cost-effective high-throughput screening of our DNA-shuffled
nanobodies with novel affinity for fuGFP.

We started our fusion protein design by searching the parts registry
for existing work on cellulose-binding domains and based our designs
on sfGFP-CBD proteins used by Imperial College’s 2014 Aqualose team.
Our results showed fuGFP-CBD can be expressed in TOP10.

Although we found that expression in TOP10 is significantly limited
in comparison to BL21(DE3), especially for three out of our four CBD
designs. From our testing, the function of fuGFP-CBDs was further
improved with the engineered addition of a flexible linker.

Most importantly, we found that fuGFP-linker-CBDcipA binds tightly
to cellulose and can be eluted using glucose to effectively purify
the protein. Ultimately, our results strongly suggest that CBD
fusion proteins can be applied in novel nanobody screening or even
adapted as a more affordable tag for protein purification using
cellulose.

**Achievements:**

* Designed fuGFP fused to 4 different CBDs
* Expressed fuGFP-CBD in both TOP10 and BL21(DE3)
* Engineering success from testing and addition of a linker to
  initial designs
* Characterised the binding and elution of fuGFP-linker-CBDcipA
* Purified fuGFP-linker-CBDcipA

**fuGFP-CBD expression in TOP10 and cellulose binding**

We cloned each fuGFP-CBD sequence into pUS250v3 and transformed them
into TOP10 *E. coli* for expression. When transformed
*E. coli* were induced with cumate on LB agar plates, we
observed green fluorescence under UV which was most clear for cells
expressing fuGFP-CBDcipA (Figure 16). Interestingly, while some
fluorescence was observed this was markedly less compared to control
cells expressing fuGFP alone which may reflect lower expression levels
of fuGFP-CBD fusion proteins in TOP10 compared to fuGFP.

![LB-kanamycin patch plates of TOP10-pUS250v3-fuGFP-CBDs. Left plate with 25 μL cumate and right plate with no cumate, each plate has four CBDs, including CBDcenA, CBDcipA, CBDcex, and CBDclos, with CBDcipA has the highest fluorescence on the 25 μL cumate plate.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure1.png)

**Figure 16.** LB-kanamycin patch plates of
TOP10-pUS250v3-fuGFP-CBDs. Kanamycin was added to 50 μg/mL
concentration and plates were incubated in 37 °C for 20 h.

We tested the binding of fuGFP-CBD proteins to cellulose using paper
filter disks and by measuring differences in fluorescence. TOP10
*E. coli* expressing each of our fusion proteins were lysed by
bead beating to obtain cell lysates containing fuGFP-CBDs. We
incubated each cell lysates with a piece of paper filter disk in a
spin column and centrifuged the column to collect the flow through. We
also washed each filter disk by adding TE buffer to the spin column
and collecting the wash fraction by centrifugation. The fluorescence
readings in Figure 17 indicate that fuGFP-CBDcipA cell lysate was the
most fluorescent and also significantly increased the fluorescence of
the filter disk in comparison to a negative empty vector control. This
suggests that out of our 4 candidate fusion protein designs,
fuGFP-CBDcipA is most easily expressed by TOP10 *E. coli* and
is functional in binding cellulose.

However, cell lysates containing fuGFP-CBD -cenA, cex, and -clos did
not have noticeable fluorescence compared to cell lysate from an empty
vector control and did not cause the filter disk to gain noticeable
fluorescence. A possible reason is that the proximity of fuGFP to the
CBD domain affects the folding of the whole fusion protein.

![A bar chart showing fluorescence readings from testing fuGFP-CBD binding to paper filter disks. Measurements taken include lysate, flow through, wash, and disc. Variables include fuGFP-cenA, fuGFP-cex, fuGFP-cipA, fuGFP-clos, and fuGFP empty vector, all with and without linker.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure2-3.png)

**Figure 17.** Fluorescence readings from testing
fuGFP-CBD (top) and fuGFP-linker-CBD (bottom) binding to paper
filter disks. 200 μL of TOP10-pUS250v3-fuGFP-CBD cell lysate was
added to a 5 mm diameter paper filter disk and incubated for 1 h
before washing with 200 μL NT buffer. Fluorescence measured in a
plate reader shows fuGFP-CBDcipA is significantly fluorescent and
causes the disk to become fluorescent. Furthermore, binding and
fluorescence is greater for fusion proteins with the engineered
linker.

**Addition of a flexible linker to improve fuGFP-CBD
functionalisation**

To investigate the low levels of fluorescence and cellulose-binding in
our initial fusion protein designs, we added a flexible 15 amino acid
glycine-serine linker between fuGFP and CBD domains. The new
fuGFP-linker-CBD sequences were again cloned into pUS250v3 and
transformed into TOP10 *E. coli* for testing.

We observed that the cell lysates obtained containing fuGFP-linker-CBD
proteins were now all brighter in fluorescence compared to a negative
empty vector control. When using the same filter paper disk binding
test, we observed that fuGFP linked to CBDcipA again causes the
greatest gain in fluorescence by the filter disk (Figure 17). However,
fuGFP linked to other CBDs (cenA, cex, clos) still did not result in
any significance fluorescence from the filter disk.

Using SDS-PAGE we analysed proteins in each sample from our filter
disk binding test. We found that after incubating the filter disk with
fuGFP-linker-CBDcipA two significant bands can be observed at around
46 kDa which corresponds to the expected size of the fuGFP-CBDcipA
fusion protein (Figure 18). A band in a similar position is observed
when the filter disk is incubated with other fuGFP-CBD fusion proteins
but is much fainter in comparison to fuGFP-CBDcipA. The presence of
these 2 separate bands may result from post-translational
modifications or protein degradation. To confirm the results, two
bands of interest for fuGFP-linker-CBDcipA were excised and analysed
with mass spectrometry which found 29% coverage of the expected amino
acid sequence and highly suggests the fusion proteins are bound to the
filter disk (Figure 19). Altogether, these results strongly suggest
that fuGFP-linker-CBDcipA is the most functional and is able to bind
cellulose and cause fluorescence.

![SDS-PAGE of samples fuGFP-linker-CBDcenA and fuGFP-linker-CBDcipA from the paper filter disk binding test. Lanes include ladder, lysate, flow through, washes, and disk samples.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure4a.png)
![SDS-PAGE of samples fuGFP-linker-CBDcex and fuGFP-linker-CBDclos from the paper filter disk binding test. Lanes include ladder, lysate, flow through, washes, and disk samples.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure4b.png)

**Figure 18.** SDS-PAGE of samples from the paper
filter disk binding test. A band at around 46 kDa is observed in
cell lysates and on the filter paper disk corresponding to the
estimated size of fusion proteins. The band of interest is most
distinct for fuGFP-linker-CBDcipA.

![Mass spectrometry results for SDS-PAGE band A in the paper filter disk binding test. Image shown 28.22% coverage of the fuGFP-CBDcipA protein sequence.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure5a.png)
![Mass spectrometry results for SDS-PAGE band A in the paper filter disk binding test. Image shown 29.70% coverage of the fuGFP-CBDcipA protein sequence.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure5b.png)

**Figure 19.** Mass spectrometry results for SDS-PAGE
bands A and B in the paper filter disk binding test shown above.
Sequencing results show that the bands of interest cover around 29%
of the expected sequence of fuGFP-linker-CBDcipA.

**fuGFP-linker-CBDcipA binds cellulose and is eluted with
glucose**

![LB-kanamycin patch plates of BL21(DE3)-pET28c(+)-fuGFP-linker-CBDs. Each plate has four CBDs, including CBDcenA, CBDcipA, CBDcex, and CBDclos.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure6.png)

**Figure 20.** LB-kanamycin patch plates of
BL21(DE3)-pET28c(+)-fuGFP-linker-CBDs. Kanamycin was added to 50
μg/mL concentration and 10 μL 0.5 M IPTG was added. Plates were
incubated at 37 °C for 20 h.

Next we tested whether fuGFP-CBD proteins bound to cellulose can be
eluted under controlled conditions. In order to increase our protein
yield, we used the BamHI and XhoI sites on each fuGFP-linker-CBD
insert to clone the fusion protein sequences from pUS250v3 into
pET28c(+) for expression in BL21(DE3). When induced with IPTG, all
transformed cells fluoresced bright green (Figure 20).

We chose to focus on fuGFP-linker-CBDcipA since previous experiments
suggested that it was the most functional. Cell lysate containing
fuGFP-linker-CBDcipA was obtained from transformed BL21(DE3) cells
induced with IPTG. Using a method replicating column chromatography,
the cell lysate was incubated with microcrystalline cellulose in a
test tube and the unbound fraction of cell lysate was removed by
centrifugation. The microcrystalline cellulose was then washed with
NT buffer which was also removed and collected by centrifugation.
Results show that fuGFP-linker-CBDcipA lysate significantly
increases the fluorescence of cellulose and is not washed off by NT
buffer (Figure 21).

![A bar chart showing fluorescence measurements of fuGFP-linker-CBDcipA bound to microcrystalline cellulose. Measurements taken include cellulose, supernatant, wash 1, wash 2, and cellulose (washed). Variables include fuGFP-linker-cipA and fuGFP control.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure7.png)

**Figure 21.** Fluorescence measurements of
fuGFP-linker-CBDcipA bound to microcrystalline cellulose. 250 μL of
cell lysate containing fuGFP-linker-CBDcipA was incubated with 250
μL of microcrystalline solution (10% w/v) for 1 h with rotation. The
supernatant was removed and the cellulose was washed twice with 250
μL of NT buffer and also removed by centrifugation each time. Bars
represent means ± SE, n = 3. Results of a Tweedie family GLM showed
a significant interaction between construct and treatment
(χ23 = 709.7, p < 0.001). Bars that don’t share a
letter are significantly different based on Tukey-Kramer post-hoc
contrast. Microcrystalline cellulose gains significant fluorescence
that is not washed off when fuGFP-linker-CBDcipA is added in
comparison to a fuGFP control.

Next, we screened elution conditions using distilled water,
cellobiose, maltose, glucose, and glycerol. After binding
fuGFP-linker-CBDcipA to microcrystalline cellulose and washing the
cellulose, we made 3 successive additions of the eluents and removed
each elution fraction by centrifugation each time. We found that with
our method, elution occurs after around 2-3 additions of eluent and
that fuGFP-linker-CBDcipA is most strongly eluted with 1 M glucose or
maltose, and weakly eluted with distilled water (Figure 22).

![A bar chart showing fluorescence measurements of elutions of fuGFP-linker-CBDcipA from microcrystalline cellulose under different conditions. Measurements taken include NT control, dH2O, 0.3 M cellobiose, 1 M maltose, 1 M glucose, and 1 M glycerol. Variables include elutions 1, 2, and 3.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure8.png)

**Figure 22.** Fluorescence measurements of elutions of
fuGFP-linker-CBDcipA from microcrystalline cellulose under different
conditions. Cell lysate containing fuGFP-linker-CBDcipA was
incubated with 250 μL of microcrystalline solution (10% w/v) for 1 h
with rotation. The supernatant was removed and the cellulose was
washed twice with 250 μL NT buffer. Fusion proteins were then eluted
from the cellulose by washing three times with 250 μL of different
conditions. Results show fluorescence of elution fractions increases
with each repetition and using 1 M glucose results in the greatest
fluorescence in the third elution.

Due to its non-fluorescent nature and ease of availability, we chose
to further examine elution using glucose. We found that glucose
elution fractions were significantly fluorescent, and this corresponds
with a decrease in the fluorescence of fuGFP-bound cellulose by
approximately half after 4 additions (Figure 23). This suggests that
glucose is an effective elution condition and could be potentially
useful in purifying fuGFP-CBDs from cellulose.

![A bar chart showing fluorescence measurements of elutions of fuGFP-linker-CBDcipA from microcrystalline cellulose using 1 M glucose. Measurements taken include cellulose (washed), elutions 1, 2, 3, and 4, and cellulose (post-elution). Variables include 1 M glucose and NT control.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure9.png)

**Figure 23.** Fluorescence measurements of elutions of
fuGFP-linker-CBDcipA from microcrystalline cellulose using 1 M
glucose. Cell lysate containing fuGFP-linker-CBDcipA was incubated
with 250 μL of microcrystalline solution (10% w/v) for 1 h with
rotation. The supernatant was removed and the cellulose was washed
twice with 250 μL NT buffer. Fusion proteins were then eluted from
the cellulose by washing four times with 250 μL of glucose (1 M).
Bars represent means ± SE, n = 3. Results of a Tweedie family GLM
showed a significant interaction between elution step and treatment
(χ23 = 3559.3, p < 0.001). Bars that don’t share a
letter are significantly different based on Tukey-Kramer post-hoc
contrast. Results show fluorescence of elution fractions increases
with each repetition and the final fluorescence of microcrystalline
cellulose with bound fusion proteins is halved at the end in
comparison to cellulose washed with only NT buffer an additional
four times as a control.

To investigate the purity of proteins obtained using glucose elution
we performed an SDS-PAGE. We found a single significant band at around
46 kDA corresponding to fuGFP-linker-CBDcipA in the elution after 3
additions of glucose (Figure 24). The identity of this band was
confirmed to be fuGFP-linker-CBDcipA via mass spectrometry which
showed 35% amino acid sequence coverage. Furthermore, this band is
also observed in the induced lysate, but is much weaker in the
supernatant and absent from the wash fractions. Moreover, the band of
interest is heavily concentrated in the cellulose sample after washing
but is decreased by around 50% after 4 rounds of glucose elution.
Densitometric analysis of the band of interest according to ImageLab
indicates fuGFP-linker-CBDcipA makes up 21% of the total protein in
induced cell lysate compared to 63% of the proteins in the cellulose
sample after incubation and washing with NT buffer, and upon elution,
fuGFP-linker-CBDcipA comprises 98% of the elution sample proteins.

Overall, these results show that fuGFP-linker-CBDcipA binds to
cellulose and can be selectively eluted using glucose. Although in our
experiment, we tested only a single glucose concentration with 4
additions and found that a good amount of fusion protein remained
bound to the cellulose. Therefore, future work could determine the
effectiveness of higher concentrations or additional rounds of elution
using glucose to increase the yield of eluted protein. Importantly, we
have shown a single band of pure fuGFP-CBD fusion protein can be
obtained which indicates that our method can be scaled-up in the
future and applied for purifying CBD-tagged fusion proteins using
cellulose.

![SDS-PAGE of fuGFP-linker-CBDcipA eluted from microcrystalline cellulose with glucose (1 M). Lanes include ladder, uninduced lysate, induced lysate, flow through, washes, elutions, and cellulose samples.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure10a.png)
![Mass spectrometry results for SDS-PAGE band C in the microcrystalline cellulose elution with glucose (1 M) test. Image shown 35.48% coverage of the fuGFP-CBDcipA protein sequence.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure10b.png)

**Figure 24.** SDS-PAGE of fuGFP-linker-CBDcipA eluted
from microcrystalline cellulose with glucose (1 M) (top). Mass
spectrometry results confirm the identity of the band outlined in
red to be fuGFP-linker-CBDcipA (bottom). The single distinct band
outlined in red is observed at around 46 kDA in the elution fraction
corresponding to our protein of interest. Furthermore, the amount of
fusion protein bound to cellulose is almost halved after 4 elutions
with glucose.

---

### Creation of fuGFP Variants

**Summary**

In response to feedback through our Human Practices work, we
endeavoured to expand the utility of the fusion proteins we had
created by generating new fluorescent proteins that are also
attached to CBDs, generating a suite of markers that can be
immobilised to and eluted from cellulose. This was done through a
degenerate primer PCR amplification. We have tested the new
fluorescent proteins - fuGFPb, fuGFPy, fuGFPa, fuBFP - and the
fluorescent protein-CBD complexes. The associated parts are
[BBa\_K4488016](http://parts.igem.org/Part%3ABBa_K4488016),
[BBa\_K4488017](http://parts.igem.org/Part%3ABBa_K4488017),
[BBa\_K4488018](http://parts.igem.org/Part%3ABBa_K4488018),
[BBa\_K4488019](http://parts.igem.org/Part%3ABBa_K4488019),
[BBa\_K4488020](http://parts.igem.org/Part%3ABBa_K4488020),
[BBa\_K4488021](http://parts.igem.org/Part%3ABBa_K4488021),
[BBa\_K4488025](http://parts.igem.org/Part%3ABBa_K4488025),
[BBa\_K4488026](http://parts.igem.org/Part%3ABBa_K4488026).

**Achievements:**

* Used PCR with degenerate primers to introduce mutations in fuGFP
  and fuGFP-linker-CBDcipA and create different coloured variants
  with different excitation and emission spectra
* Showed that different coloured variants of fusion proteins can
  bind cellulose
* Showed that different coloured variants are eluted from cellulose
  with glucose
* Purified different coloured variants of fuGFP-linker-CBDcipA

**Generation of Novel fuGFP variants**

![images of fuGFP variants in pUS252, transformed into TOP10 E. coli and displayed on an agar plate. The plate shows the four different variant colours of fuGFP. Visually it resembles a galaxy spotted with stars, but the stars are florescent microbes!](https://static.igem.wiki/teams/4488/wiki/photos/results-var-figure25.jpg)

**Figure 25.** fuGFP variants in pUS252, transformed
into TOP10 *E. coli*. Plate shows the four different
variant colours of fuGFP.

Five new fluorescent free-use proteins were generated using our
degenerate primer protocol. All five were characterised in the plasmid
pUS252, transformed into TOP10 *E. coli*. Spectral fluorescence
data was measured, as seen in Figure 26 below. Based on this data,
four out of five new fluorescent free-use proteins were selected to be
uploaded as new parts (both basic and composite parts, attached to our
cellulose-binding domains). fuBFPt (t for threonine), was not
included, as it is almost identical in spectra to fuBFP, however
fluoresces at a much lower intensity.

![Excitation/Emission spectra for the variants of fuGFPs (fuGFPy, fuGFPb, fuGFPa).](https://static.igem.wiki/teams/4488/wiki/photos/results-var-figure1.png)

**Figure 26.** Excitation/Emission spectra for the
variants of fuGFPs. Measurement settings for the plate reader can be
found under
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).
Fluorescence intensity has been normalised so that all values are
between 0 and 1 to reduce the measurement and sample artefacts. Four
distinct fuGFPs are visualised. The original fuGFP is shown in
purple, and is excited in the long wave UV range. The other three,
fuGFPy, fuGFPb and fuGFPa, are all excited in the visible spectrum.
fuGFPb (green lines on the plot) has a maximum excitation wavelength
of 485 nm. fuGFPy (yellow lines on the plot) has a maximum
excitation wavelength of 488 nm. fuGFPa (fuGFP-alanine, blue lines
on the plot) has a maximum excitation wavelength of 482 nm.

fuGFPb

fuGFPb is a blue-shifted variant of fuGFP. Unlike fuGFP, which is
excited in the long-wave UV range, fuGFPb is excited by blue light.
This makes fuGFPb compatible with plate readers that do not extend
into the UV range for excitation, and adds utility to the free-use
protein line as analogues for sfGFP, which does fluoresce under blue
light. fuGFPb has a maximum excitation wavelength of around 485 nm.

fuGFPy

fuGFPy is a yellow-shifted variant of fuGFP that appears slightly more
yellow in its fluorescence. Similar to fuGFPb, fuGFPy is excited by
blue light, however its maximum excitation wavelength is around 488
nm.

fuGFPa

fuGFPa (a for alanine, referring to its mutant amino acid), is a
blue-shifted variant of fuGFP. It appears slightly more yellow/orange
than fuGFPy on a plate, although the reasons for this are currently
unclear. Both its excitation and emission spectra are more blue
shifted than fuGFPb and fuGFPy. fuGFPa has a weaker fluorescence
profile than fuGFPy and fuGFPb. Its maximum excitation wavelength is
around 482 nm.

![Excitation/Emission Spectra for fuBFPs.](https://static.igem.wiki/teams/4488/wiki/photos/results-var-figure2a.png)

**Figure 27a.** Excitation/Emission spectra for fuBFPs.
Measurement settings for the plate reader can be found under
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).
Fluorescence intensity has been normalised so that all values are
between 0 and 1 to reduce the measurement and sample artefacts. 2
distinct fuBFPs (fuBFP and fuBFPt) have been visualised.

![Excitation/Emission Spectra for fuBFPs.](https://static.igem.wiki/teams/4488/wiki/photos/results-var-figure2b-1.png)

**Figure 27b.** Excitation/Emission spectra for fuBFPs.
Measurement settings for the plate reader can be found under
[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).
**(b)** has the raw values where fluorescence intensity
has not been normalised. 2 distinct fuBFPs (fuBFP and fuBFPt) have
been visualised. While the spectra for both fuBFPs are remarkably
similar, fuBFPt is far weaker in fluorescence intensity.

fuBFP

fuBFP (free-use blue fluorescent protein) is one of two BFPs
generated. The other, fuBFPt (t for threonine, referring to its mutant
amino acid) fluoresces only very faintly compared to fuBFP. fuBFP
fluoresces in the long wave UV range, similar to fuGFP. Its maximum
excitation wavelength is around 377 nm (Figure 27).

**fuGFP-CBD Variants**

One potential application of improved fuGFP with different colour
variants is that they can be fused to CBDs and immobilised onto
cellulose. The different colour variants could provide a basis for the
design of a lateral flow assay that detects multiple analytes. Using
the degenerate primer MVS70 (with redundancy at three loci of the
fluorophore) we amplified plasmids of pET28-fuGFP-linker-CBDs to
generate variant fluorescent proteins which we transformed into
BL21(DE3) using the heat-shock method. We induced protein expression
in selected colonies of BL21(DE3) and obtained lysates containing
variant fuGFP-CBDs. We then tested the function of these variants by
binding to microcrystalline and eluting with glucose.

Using the plate reader to measure the fluorescence of samples of
eluted protein from cellulose using glucose. We identified the
variants of fuGFP-CBDs that we created and characterised their
excitation and emission spectra (Figure 28). Subtle differences exist,
fuGFP-linker-CBDcipA excites maximally at around 395 nm and has
maximum emission at around 510 nm. In comparison fuGFPb-linker-CBDcipA
excites maximally at ~485 nm and emits maximally at ~510 nm.
fuGFPy-linker-CBDcipA excites maximally at around 490 nm while
emitting maximally at around 510 nm. fuGFPa-linker-CBDcipA excites
maximally at 480 nm and emits maximally at ~510 nm. Finally,
fuBFP-linker-CBDcipA excites maximally at around 380 nm and emits
maximally at around 450 nm.

![A line graph showing excitation and emission spectra measured from fuGFP-linker-CBDcipA, fuGFPy-linker-CBDcipA, and fuBFP-linker-CBDcipA.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure11.png)

**Figure 28.** Excitation/Emission spectra measured
from fuGFP-, fuGFPb-, fuGFPy-, fuGFPa-, fuBFP-, linked to CBDcipA.
Samples of the fuGFP-, fuGFPy-, fuBFP-fusion proteins were eluted
with glucose after binding to cellulose and their spectra were
measured in a plate reader using our plate reader protocol. Samples
of fuGFPb- and fuGFPa- fusion proteins were measured from cell
lysates.

Furthermore, we tested the ability of these GFP-linker-CBDcipA
variants to bind to microcrystalline cellulose and their elution under
glucose. Results in Figure 29 show fuGFP-linker-CBDcipA and its
variants are functional and bind to cellulose resulting in increased
fluorescence detected by the plate reader and can each be eluted with
1 M glucose.

![Cellulose binding test for fuGFP-linker-CBDcipA. Measurements taken include lysate, cellulose (bound), cellulose (washed), cellulose (post-elution), washes 1, 2, 3, elutions 1, 2, 3, and 4.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure12a.png)
![Cellulose binding test for fuGFPy-linker-CBDcipA. Measurements taken include lysate, cellulose (bound), cellulose (washed), cellulose (post-elution), washes 1, 2, 3, elutions 1, 2, 3, and 4.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure12b.png)
![Cellulose binding test for fuBFP-linker-CBDcipA. Measurements taken include lysate, cellulose (bound), cellulose (washed), cellulose (post-elution), washes 1, 2, 3, elutions 1, 2, 3, and 4.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure12c.png)

**Figure 29.** Cellulose binding test for colour
variants of GFP-linker-CBDcipA. 250 μL of lysate containing each of
the three colour variants was incubated with 250 μL of
microcrystalline cellulose (0.1 g/mL) for 1 hour with mixing. The
cellulose was washed with 250 μL NT buffer twice followed by the
addition of 250 μL glucose (1 M) twice to elute the proteins. Bars
represent means ± SE, n = 2. Results of Gaussian family GLMs showed
a significant difference between treatments for all variants. Bars
not sharing the same letter are significantly different based on a
Tukey-Kramer post-hoc test.

SDS-PAGE of samples eluted from cellulose using glucose shows distinct
single bands for each colour variant fusion protein at ~46 kDA
corresponding to the expected sizes (Figure 30). These results show
that the colour variants of fuGFP-linker-CBDcipA bind to cellulose and
can be eluted with glucose to obtain fractions with relatively high
purity.

![SDS-PAGE gel comparing lysate and elution samples for each colour variant of fuGFP-linker-CBDcipA.](https://static.igem.wiki/teams/4488/wiki/photos/results-cbd-figure13.png)

**Figure 30.** SDS-PAGE gel comparing lysate and
elution samples for each colour variant of fuGFP-linker-CBDcipA.
Lysate was obtained from bead beating BL21(DE3) cells expressing the
proteins of interest. Elution samples were obtained by washing
microcrystalline cellulose with bound proteins using glucose (1 M).
Distinct single bands at ~46 kDa are observed in lanes 4, 6, and 8
corresponding to fuGFP-, fuGFPy-, and fuBFP- respectively fused to
CBDcipA by a linker.

---

### Basic Part Characterisation - fuGFP

Free-use GFP (fuGFP) was designed by Mark Somerville at the University
of Sydney with the aim of creating an unpatented GFP variant that
would facilitate any kind of synthetic biology research by any
organisation. The part ([BBa\_K3814004](http://parts.igem.org/Part%3ABBa_K3814004)) was added to the iGEM parts registry by the 2021 University of
Sydney Team.

Our team has contributed to the characterisation of this part by
calculating a theoretical absorbance spectrum (Figure 31) for a 1
mg/ml concentration solution of fuGFP from experimental data,
enabling future teams to use spectroscopy to quantify the
concentration of this part.

![Image of theoretical absorbance spectra for fuGFP and sfGFP. sfGFP shows an absorbance peak exceeding 1.5 in the range of approximately 475-500 nanometers, while fuGFP shows an absorbance peak at 1 in the wavelength range of 390-400 nanometers.](https://static.igem.wiki/teams/4488/wiki/photos/contributions-fusfgfp.png)

**Figure 31.** Theoretical absorption spectra of
fuGFP and sfGFP at 1 mg/ml between 335-550 nm.

The absorbances were measured in the photospectrometer between 300-700
nm and the average was graphed (Figure 31). We found that fuGFP
exhibits peak absorbance at 395 nm with a secondary peak at 475 nm. In
comparison, sfGFP displays only a single peak at 490 nm.

We obtained purified fuGFP and sfGFP stock solutions from Mark
Somerville and made 1:5 and 1:8 dilutions of both proteins. In
addition to these concentrations, we made up a 1:9 dilution in 0.2 M
NaOH. This deprotonates the chromophore of the fluoroproteins such
that they take on a homologous conformation between sfGFP and fuGFP,
resulting in identical absorption spectra at these wavelengths (Ward
*et al.* 1981). The extinction coefficient of GFPs with this
denatured chromophore at 446 nm is 44,000 M−1 cm−1
(Bomati *et al.* 2015).

The Beer-Lambert law was used to back calculate the concentrations of
the original solutions, which were determined to be 1.5 mg/ml and 1.4
mg/ml for fuGFP and sfGFP respectively. From these concentrations, the
extinction coefficient of fuGFP was calculated to be 27618 M−1
cm−1. To normalise the absorption values for a 1 mg/ml
solution, we divided the absorption values by their respective
concentrations and graphed the resulting spectra (Figure 31).

![Image of Excitation (340 to 520 nanometers) and Emission (480 to 560 nanometers) spectra of fuGFP and sfGFP. it shows that while emission peaks are similar for the two GFP spectra, fuGFP excites with light of a lower wavelength (about 400 nm) than sfGFP (about 500 nm).](https://static.igem.wiki/teams/4488/wiki/photos/contribution-es-em-fu-sf.png)

**Figure 32.** Excitation (340-520 nm) and Emission
(480-560 nm) spectra of fuGFP and sfGFP.

Excitation and emission spectra of 200 µL from the same dilutions and
stock solutions were measured using the plate reader. Excitation
values were measured from 340-520 nm and emission values were measured
from 480-560 nm for both sfGFP and fuGFP and normalised against their
peak RFU values (Figure 32).

---

**References**

Bomati, E.K., Haley, J.E., Noel, J.P., and Deheyn, D.D., 2015. Spectral
and structural comparison between bright and dim green fluorescent
proteins in Amphioxus. *Scientific Reports*, *4*(1).

Brooks, M. E., Kristensen, K., van Benthem, K. J., Magnusson, A., Berg, C.
W., Nielsen, A., Skaug, H.J., Maechler, M., Bolker, B.M., 2017. “glmmTMB
Balances Speed and Flexibility Among Packages for Zero-inflated
Generalized Linear Mixed Modeling.” *The R Journal*, *9*(2),
pp.378-400.

Fox, J., Weisberg, S., 2019. An R Companion to Applied Regression, Third
edition. Sage, Thousand Oaks, CA.
<https://socialsciences.mcmaster.ca/jfox/Books/Companion/>

Hartig, F., 2022. DHARMa: Residual Diagnostics for Hierarchical
(Multi-Level / Mixed) Regression Models\_. R package version 0.4.6,
[https://CRAN.R-project.org/package=DHARMa](https://CRAN.R-project.org/package%3DDHARMa)

Lenth, R., 2022. emmeans: Estimated Marginal Means, aka Least-Squares
Means. R package version 1.7.4-1.
[https://CRAN.R-project.org/package=emmeans](https://CRAN.R-project.org/package%3Demmeans)

R Core Team, 2022. R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria.
<https://www.R-project.org/>

Ward, W., DeLuca, M. and McElroy, W., 1981.
*PROPERTIES OF THE COELENTERATE GREEN-FLUORESCENT PROTEINS -
Bioluminescence and Chemiluminescence.*
1st ed. Saint Louis: Elsevier Science, pp.235–242.

{% endblock %}