iGEM 2023 — Team anu-australia

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Here's everything we've done on our project day by day.{% endblock %}
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###### 28/06/2023

1. PDB pymol images were examined for a template to run through the neural networks pMPNN and RFDiffusion
2. 6J05.pdb was decided on due to it having all the binding site on the one monomer rather than spread between two dimers

![](https://static.igem.wiki/teams/4780/wiki/comp-protein.webp)

###### 29/06/2023

1. Interface residues were identified between the monomers of the ArsR template 6J05.pdb using the pymol command

1. InterfaceResidues 6J05, chain A, chain B)

2. Interface residues were specified using the pymol script

1. iterate (chain A and interface and name CA), print(resi)

3. This produced the list of important interfacial residues between the monomers that would need to be held fixed before being run through the neural network in order to increase the likelihood of dimerisation and therefore binding stability
4. 7, 10, 11, 14, 15, 17, 18, 19, 20, 23, 26, 27, 30, 81, 84, 85, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 105
5. The ligand binding site residues were indentified as amino acids 5 angstroms or less from the Arsenic
6. These residues were as follows; 95, 96, 100, 101, 102, 104.
7. Therefore total number of fixed residues for the protein before being run through protein MPNN were as follows;

1. 7, 10, 11, 14, 15, 17, 18, 19, 20, 23, 26, 27, 30, 33, 81, 84, 85, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105

8. [ProteinMPNN model](https://huggingface.co/spaces/simonduerr/ProteinMPNN)
9. The 6J05.pdb was run through the protein MPNN online hugging face interface with all previously held residues fixed in order to produce 10 output amino acid sequences

###### 30/06/2023

**De novo protein design method:**

1. New RFDiffusion interface was used for de novo protein design link below

1. [Protein Generation via Diffusion in Sequence Space](https://huggingface.co/spaces/merle/PROTEIN_GENERATOR)
2. Input: 6J05 length (105 amino acids long)

**Active site residues were indentified in the previous day**1. 95C, 96C, 100R, 101D, 102C, 104L
2. In between residues to make one continuous range since, the interface didn't accept discontinuous binding site fragments
3. 95C, 96C, 97H, 98G, 99T, 100R, 101D, 102C, 103A, 104L
4. Without amino acids: aa95-104

2. Four versions of the RFDiffusion models were made, each with slightly different conditions as specified below.

**Version (1)**1. Therefore, to make similar to WT 6J05 94 amino acids before the motif and 1 amino acid after the motif. **(3 iterations)**

“94,A95-104,1”
**Version (2)**2. Including the amino acid at 105 from WT structure **(3 iterations)**

“94,A95-105”
**Version (3)**3. With a balance of amino acids either side **(3 iterations)**

“48,A95-104,48
**Version (4)**4. With a balance of amino acids either side (motif 95-105) **(3 iterations)**

“48,A95-105,48”
3. Version 1 and 3 contained the binding motif as specified by the pymol commands
4. Version 2 and 4 contained one extra amino acid as part of the fixed binding motif - given the binding motif was at the end of the protein, it might be interesting to specifiy the entire tail binding motif.
5. Version 1 and 2 put the binding motif at the end of the protein as is found in the WT ArsR template.
6. Version 3 and 4 put the binding motif in the centre of the protein to assess if there was any difference in binding ability
7. 3 iterations were made for each version and the highest ranking pLDDT was chosen to be ordered

![](https://static.igem.wiki/teams/4780/wiki/notebook-anthony-in-freezer1.jpg)

![](https://static.igem.wiki/teams/4780/wiki/notebook-anthony-fiddling-with-gel-min.jpg)

###### 15/08/2023

1. Inocculation of LB broth with ampicillin and cells (BL21(DE3)+pET21b)
2. Dilution of gBlocks (gene fragments) to 25 ng/uL in water

###### 16/08/2023

1. Miniprep of (BL21)(DE3)+pET21b plasmids as backbone for fragment cloning
2. Restriction enzyme NdeI and XhoI NDE1 reacted with pET21b plasmid to prepare plasmid for cloning
3. Cultured phi29 (Host: 5547, Bacillus subtilis phage)

###### 22/08/2023

1. Purification of pet21b backbone by gel electrophoresis

1. Expected 2 bands, observed 3+ (1.6 Kb, 5.4 Kb, 7 Kb)
2. Band at 5.4 Kb isolated

2. Purification of pet21b backbone by centrifugation (Wizard SV Gel and PCR Clean-up System)

###### 23/08/2023

1. Infused insert with backbone and enzyme and incubated for 15 mins at 50 degrees
2. Transformed cloned insert fragment into competent stella cells and plated out onto ampicillin agar. Transformations were named as follows (and these are the number codes we use throughout the rest of our project):

|  |  |  |
| --- | --- | --- |
| 1. ArsR E. coli MX 2. AfArsR MPNN1 MX 3. MPNN2 MX 4. MPNN3 MX 5. MPNN4 MX 6. MPNN5 MX 7. MPNN6 MX 8. MPNN7 MX 9. MPNN8 MX 10. MPNN9 MX 11. MPNN10 MX 12. RfDiffusion 4.1 MX 13. RfDiffusion 2.1 QT | 14. RfDiffusion 2.1 MX 15. ArsR C. glutamicum QT 16. ArsR A. ferrooxidans QT 17. ArsR E. coli QT 18. AfArsR MPNN1 QT 19. MPNN2 QT 20. MPNN3 QT 21. MPNN4 QT 22. MPNN5 QT 23. MPNN6 QT 24. MPNN7 QT 25. MPNN8 QT 26. MPNN9 QT | 27. MPNN10 QT 28. RFDiffusion 4.1 QT 29. ArsR C. glutamicum MX 30. ArsR A. ferooxidans MX 31. QTEncapsulin 32. Type 1 EncA 33. RfDiffusion 3.3 MX 34. RfDiffusion 3.3 QT 35. RFDiffusion 1.2 36. Metallothionein F MX 37. MT2 MX 38. MT2 QT (potentially MT1 QT) |

3. Harvested phi29 (Host: 5547, Bacillus subtilis phage)

1. Phage lysate stored in 4 degree fridge

###### 25/08/2023

1. Titrated phi29 cell lysate (Host: 5547, Bacillus subtilis phage)
2. Cultured vB\_bSup\_Goe1 (Host: 402, Bacillus subtilis phage)

###### 28/08/2023

![](https://static.igem.wiki/teams/4780/wiki/notebook-sabrina-s-phage-plaques-min.jpg)

1. Innoculating overnight cultures for transformants 1-18: Pick colony from each plate and innoculate 5 mL LBAmp

###### 29/08/2023

1. Innoculating overnight cultures for transformants 19-34: Pick colony from each plate and innoculate 5 mL LBAmp
2. Miniprep transformants 1-18: Follow miniprep protocol

###### 30/08/2023

1. Miniprep transformants 19-35: Follow miniprep protocol
2. Minipreped sequences for all variants sent off for sequencing
3. Re-cultured vB\_bSup\_Goe1 (Host: 402, B. subtilis phage): used 200 uL of phage lysate supplied instead of 100 uL (observed poor phage growth from first culture)
4. Counted phi29 titration experiment: Current phi29 lysate solution has phage at 4 x 106 PFU/mL (4 degree fridge)

###### 01/09/2023

1. Harvested phage vB\_bSup\_Goe1 (Host: 402, B. subtilis phage): stored in 4 degree fridge
2. Titrated vB\_bSup\_Goe1 phage lysate: used 200 uL (instead of 100 uL) of 402 B. subtilis for plate culture

###### 04/09/2023

1. Analysed sequencing: chromatograms really messy, some have the target gene but still not good enough to use. We think that the digestion is where it went wrong so must redo.
2. Dilute cas9 forward and reverse primers to 1ng/100uL
3. Inocculation of LB broth with ampicillin and cells (BL21(DE3)+pET21b)

###### 05/09/2023

1. Miniprep of (BL21)(DE3)+pET21b plasmids as backbone for fragment cloning ("mini pet21" eppendorfs)
2. Digestion: Restriction enzyme NdeI and XhoI NDE1 reacted with pET21b plasmid to prepare plasmid for cloning

1. backbone digested for an extra hour on the PCR machine

###### 07/09/2023

1. Infusion cloning: Very low digested vector concentration (~3ng/µl), so used 7µl plasmid, 1µl insert, 2µl mastermix.
2. Transformation: Heat shock into stellar E. coli cells. Incubation at 37 °C overnight.

###### 11/09/2023

1. Inoculations of 1-4, 17-20, 31-34 into 5 mL LB Amp, O/N incubation at 37 °C.
2. Very low transformation rate of 33. ~10 colonies compared to 100s for other transformants.

###### 13/09/2023

1. Inoculation of LB broth containing kanamycin with E. coli (containing pRH030 plasmid).
2. Miniprep of pRH030 plasmids fpr use as backbone for phage gene inserts

###### 19/09/2023

![](https://static.igem.wiki/teams/4780/wiki/exp-19-09.jpg)

1. Annealing guide RNA at 95°C for 5 hours and ramping down to 4 °C
2. Gel purification of digested CRISPR backbone

###### 22/09/2023

1. Second attempt at ligating gRNAs with CRISPR/Cas9 backbone

1. Ratio of plasmid:guide:enzyme in ligation = 3:5:2

2. Transformed ligated DNA into E. coli

###### 26/09/2023

1. Growing protein cultures (31.2, 20.1, 17.2)

1. Optimise IPTG induction with 2 diff temperatures (20 °C, 37 °C) and 3 diff IPTG concentrations (0 mM, 0.5 mM, 1.0 mM)
2. Find best OD and use it's conditions

###### 27/09/2023

![](https://static.igem.wiki/teams/4780/wiki/notebook-riley-on-sds-page-min.jpg)

1. Expressed small scale cultures were run on an SDS-PAGE gel to assess which expression conditions was best

1. The gel didn't show any convincing expression bands - however large scale cultures went ahead due to the time constraints before the final showcase

2. Glycerol stocks were made for all the recloned samples refer to transformation plate ID:
3. 400 mL large scale cultures were run for 17.1 20.1 and 31 and induced at OD~0.6 with 1mM of IPTG after ~3 hours of incubation
4. Large scale cultures were left to express overnight for purification with the AKTA

###### 28/09/2023

1. Proteins 17.1, 20.1, and 31.2 were purified with the AKTA
2. SDS page of proteins 17.1 and 20.1 showed no protein expression

![](https://static.igem.wiki/teams/4780/wiki/group-photo-at-synbio-min.webp)

###### 29/09/2023

We presented our work at the Australasian Synthetic Biology Challenge final showcase alonside 6 other Australian synthetic biology teams.

###### 03/10/2023

![](https://static.igem.wiki/teams/4780/wiki/notebook-glycerol-stock.jpg)

1. Innoculations of 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2 from glycerol stocks into 5 mL LB Amp
2. SDS PAGE of 20 (QT Enc) at different stages of NiNTA purification
3. Heat shock transformation of 17.1, 20.2, 31.1 into Shuffle T7 Express cells, culture overnight on LB Amp plates
4. Agar gel seperation of digested pET21-GST vector

###### 04/10/2023

1. Native page is running and should be done tomorrow morning (gel; ladder, A11, A12, B1)
2. Metallothioneins (36,37,38) and other ArsR variants (15,16,29,30) were cloned and transformed into cells which were plated out - colonies will need to be picked tomorrow and LB cultures inoculated
3. Shuffle cells were picked and inoculated into LB Kan 6 mL and grown overnight
4. Miniprepped gRNA plasmids from previous round of transformation (round 2)

###### 05/10/2023

**Things to do**

1. Stain Native PAGE
2. Metallothioneins (LBK) and AsrR (LBA) variants picked and inoculated into LB culture overnight
3. Shuffle cells (17.1, 20.2, 31.1) and Glycerol stock (18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) primary cultures transfered (100 μL) to secondary cultures (5 mL) and monitored for OD=0.6 for induction by IPTG

![](https://static.igem.wiki/teams/4780/wiki/notebook-encapsulin-native-unsuccessful-min.jpg)

**Completed**

1. Native PAGE stained - no bands at top of gel unfortunately
2. Shuffle cells (17.1, 20.2, 31.1) and glycerol stocks (18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) were transfered to secondary cultures and induced at OD ~0.4-0.6
3. Metallothioneins (LBK) (36, 37, 38) and AsrR (LBA) (15, 16, 29, 30) were picked and inoculated as triplicates overnight (labelled "LBA/K Infusion 36.2"...etc)
4. Transformed and plated of Stellar E. coli with gRNA Cas9 plasmids (round 3)

1. Used 2x concentrated gRNA, pRH030 plasmid, ligase, water (diluent) in ratio of 3:3:2:2

###### 06/10/2023

![](https://static.igem.wiki/teams/4780/wiki/notebook-riley-and-jasper-pipetting-min.jpg)

**Things to do**

1. Lyse and miniprep Metallothioneins and ArsR triplicates for sanger sequencing
2. Lyse and denature shuffle cells (17.1, 20.2, 31.1) and Glycerol stock (18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) for a run on an SDS-PAGE gel to assess protein expression
3. Miniprep successfully transformed Stellar cells with gRNA Cas9 plasmids

**Completed**

1. Submitted miniprepped gRNA plasmids (501, 512, 785 - round 2) for Sanger sequencing
2. Shuffle cells and glycerol stocks (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) run on SDS gels and left to destain over weekend

###### 10/10/2023

**Things to do**

1. Check destained SDS gels of (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2)
2. Try a Western Blot of (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2), it is likely the protein is not overexpressed enough to see results on the SDS gel
3. Inoculate 5mL primary cultures of all the miniprepped DNA after transformation into Stella cells
4. Set up Native-PAGE
5. Maybe make glycerol stocks for Stella cells (17.1, 20.2, 31.1)

![](https://static.igem.wiki/teams/4780/wiki/notebook-mbp-and-encapsulin-good-gel-min.jpg)

**Completed**

1. photographed destained SDS gels of (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) from Friday
2. Inocculate 5ml cultures of gRNA Stellar cells (500, 502, 513, 515 x 2, %1 x 3, 785 x 3, 147 x 3 ...) with Kanamycin
3. Native PAGE of Encapsulins

1. Ladder - 17.1 (shuffle)- 31.1 (shuffle) - 32.1 (BL21) --- 17.1 - 31.1 - 32.1.
2. First lot 15 microlitre, second 5 microlitre.
3. Samples sonicated, then centrifuged. Samples did get warm from sonication.

4. Transformations of Metallothionein +Enc, metallothionein, some arsR, some de novo into shuffle T7/shuffle T7 express cells

1. Shuffle T7 Express cells: 31.1 + 38.2, 32.2 + 36.2, 32.2 + 37.1, 36.2, 37.1 (combinations transformed with ampicillin and kanamycin, singles transformed with just kanamycin)
2. Shuffle T7 cells: 13.2, 15.3, 16.1, 19.1, 20.2 (all transformed with ampicillin)

###### 11/10/2023

**Things to do**

1. Stain Native-PAGE
2. Inoculate Primary cultures into 5mL cultures the morning of all successfully transformed cells
3. 20.2 and 36.2 failed (will re-transform with 38.2 later today)
4. Maybe make glycerol stocks for stella cells (17.1, 20.2, 31.1)

**Completed**

1. Primary cultures were made for the cells that successfully transformed (13.2, 15.3, 16.1, 19.1, 37.1)
2. Native-PAGE is being destained
3. Re-transformed 36.2, 38.2, 29.3, 30.3 and co-transformed 31.1 + 38.2 and 32.2 + 37.1

###### 12/10/2023

**Things to do**

1. Inoculate primary 5 mL cultures of re-transformed/ co-transformed plates (36.2, 38.2, 29.3, 30.3, 31.1 + 38.2 and 32.2 + 37.1)
2. Photograph destained Native-PAGE

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
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#### Expression of cysteine-rich binding proteins

With the modular nature of our project, we had many moving parts which required extensive engineering, from plasmid design
to phage engineering. One of these components was expression of our metal-binding proteins and encapsulins in an *E. coli*
model system. To first optimise expression of each of these proteins, we aimed to express them individually. In our first
iteration of this component, we designed and cloned our plasmids containing genes encoding various metal-binding proteins
and encapsulins, a process of its own, and transformed BL21(DE3) *E. coli* cells. However, we were unable to isolate our target
proteins from these transformed cells nor confirm overexpression. Considering our metal binding proteins contain high levels
of cysteine residues which may complicate their folding and stability in a non-specialised host, we considered whether using
a different *E. coli* strain better suited to expression of such high-cysteine proteins might yield better results. Learning
from our first cycle, we decided to try transforming Shuffle *E. coli* cells, which are characterised by their ability to
express correctly-folded high-cysteine proteins1. In this second cycle, our protein expression was more successful. In light
of these results, we proceeded with a third cycle of engineering, this time aiming to co-express metal-binding proteins and
encapsulins.

#### gRNA Design

Another component of our system involves using engineering phages to deliver genes encoding metal-binding proteins and
encapsulins into bacteria. An important component to this arm of our project was the design of an efficient CRISPR/Cas9
system which could select for recombinant phages. However, we did not know which genes in our chosen phages were critical
to their replication. Guided by literature on other *B. subtilis* phages, we designed guide RNAs (gRNAs) targeting various
hypothetical proteins in the Φ29 genome. We tried to clone our designed gRNAs into a Cas9 plasmid, but this step posed an
unexpected challenge. In our first attempt at an infusion transformation to construct our plasmid, we observed no transformed
colonies. We learned that issues with infusion transformation could occur when using very short fragments of nucleic acids,
such as our gRNAs. In our second cycle of transformation, we tried to optimise our protocol, increasing the ratio of gRNA.
We observed some transformed colonies, but still very little success overall. Sequencing plasmids from these transformed
colonies, we found they did contain the target plasmid, indicating the issue in our previous protocol was indeed the low
ratio of gRNA. Utilising this information, we went through a third cycle of infusion transformation where we further
increased the ratio of gRNA which resulted in much more success, allowing us to move on to the next step of testing our
gRNAs.

#### References

1. Zhang, X., Nguyen, N., Breen, S., Outram, M. A., Dodds, P. N., Kobe, B., Solomon, P. S. & Williams, S. J. 2017.
   Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies. Molecular
   Plant Pathology, 18 (1), 141-151.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
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## Experimental Overview

The experimental workflow can be divided into two distinct concurrent projects.
The first involves the engineering of the encapsulin-MBP system which will be
responsible for the bioaccumulation of arsenic. This system is designed to be modular,
with all components contained on one operon meaning Golden Gate cloning can easily
be applied to substitute in various encapsulins and MBPs to optimise the system for
different organisms and environments. The second involves designing a phage delivery
system. This allows for targeted expression of our construct within a chosen
environment, which can easily be modified to target different species without the
need to genetically modify the species directly.

To increase our chance of successful gene expression we have chosen four species to
apply our platform to, with up to four phages for each species. We have selected two
model species, *B. subtilis* (gram positive) and *P. putida* (gram negative); and two
species more representative of species found in pollution sites, *C. glutamicium*
(gram positive) and *V. natriegens* (gram negative). *E. coli* will also be used to
produce proteins for purification and testing of protein properties such as binding
and localisation to encapsulin.

### Protein Engineering

Before translating this system to specialist bacteria commonly found in heavy-metal
polluted sites, we will use recombinant expression in *E. coli* as a proof of concept.
The formation of nanocompartments and their capacity to internalise metal binding
proteins (MBPs) will be assessed using native PAGE, negative stain transmission
electron microscopy (TEM), and isothermal titration calorimetry (ITC). Using our
control constructs, ITC and TEM in particular will provide
information regarding the degree to which these nanocompartments can internalise
heavy metals, where we will use antimony as an arsenic analogue for these experiments.
After this characterization, the most promising encapsulins and MBPs will be
transformed into specialist alkaliphilic bacteria using a phage-based delivery
platform, and expressed under species-specific regulatory elements.

### Bacteriophage Engineering

To get stable expression of our constructs within bacteria we have decided to use
lytic phages, preventing the virus from going into a lysogenic phase and becoming
dormant. We have identified multiple candidate phages for each bacterial species of
interest, and will test which phages are best at expressing foreign proteins by using
GFP as a marker gene for successful expression. This will allow for the phage
expression system to be developed before our encapsulin-MBP operons are ready for
expression. Our gene constructs will be inserted into the structure and assembly
portion of the viral genome through homologous recombination, and successful clones
selected using Cas13 counterselection. This will allow us to select against phages
without successful insertion of our gene. Further modification of phages such as
removal of lysis genes, or modification of structural proteins may be pursued to
optimise properties such as viral specificity and bioaccumulation capacity.

## Protocols

###### Wizard SV Gel and PCR Clean-Up System

###### DNA Purification by Centrifugation

###### Gel Slice and PCR Product Preparation

**A. Dissolving the Gel Slice**

1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5mL centrifuge tube.

2. dd 10 μL Membrane Binding Solution per 10mg of gel slice. Mix and incubate at 50-65°C until gel slice is completely dissolved.

**B. Binding of DNA**

3. Insert SV Minicolumn into Collection Tube.

4. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.

5. Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

**C. Washing**

6. Add 700μL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

7. Repeat Step 4 with 500μL Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes.

8. Empty the Collection Tube and centrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

**D. Elution**

9. Carefully transfer Minicolumn to a clean 1.5mL microcentrifuge tube.

10. Add 50μL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 x g for 1 minute twice

11. Discard Minicolumn and store DNA at 4°C

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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As can be seen from the protein expression SDS-PAGE, although much of the cell lysis on the
gel impairs any discernment of exact protein expression, there appears to be an overexpression
band at around the 35 kDa mark. This is likely evidence of the expression of our encapsulin
monomer under reducing conditions. To assess the possibility of cellular expression of
encapsulins, future non-reducing Native-PAGE gels are being conducted to visualize the
assembling capacity of the nanocompartment.

![](https://static.igem.wiki/teams/4780/wiki/results-labelled-gel1.png)

**Successful gel displaying overexpression of encapsulin**

#### Future directions

Future directions for our project will likely focus on producing assembled
encapsulin nanocages containing both natural and engineered metal binding proteins at large
scales for biochemical and structural characterization. An open question in encapsulin
research is the efficiency of which heterologous cargo can be targeted to the interior
of the protein nanocage, which may have implications for the efficiency of our system.
To assess the degree to which our metal-binding proteins are encapsulated, negative stain
transmission electron microscopy or cryogenic electron microscopy of purified nanocages
could be used. In future designs, the sequence of the targeting peptide appended to the
cargo proteins may also be varied to or from the consensus sequence to manipulate the
degree of cargo internalization accordingly.

While preliminary results suggest that metal-binding proteins produced
engineered using ProteinMPNN may have improved soluble expression compared to the wild type,
it is uncertain as to whether they retain their native metal binding function. To ascertain
this, we can employ differential scanning fluorimetry (DSF) to gain information about the
kinetics of metal binding. An alternative approach that could make use of the advanced
joint mass spectrometry facilities at the ANU is native mass spectrometry, which may allow
us to better resolve the metal binding dynamics of our engineered variants in their native
state. This method may also have greater utility for evaluating the binding capabilities
of these proteins once internalized within the encapsulin.

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