iGEM 2024 — Team ualberta


{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}An outline of the engineering cycles we completed throughout ReneWool’s development{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5427/engineering-success-assets/engineering-success-headerv2.webp)

# Engineering Success

An outline of the engineering cycles we completed throughout ReneWool’s development

[Project Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Future Directions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27future%27%29%20%7D%7D)
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

[ ]

[Engineering The Inducible URS Series pTac\_RBS 1/2/3/4](#Engineering-The-Inducible-URS-series-pTac_RBS-1/2/3/4)
 [Engineering Process for Spider Silk](#Engineering-Process-for-Spider-Silk)
 [Engineering The Constitutive URS J23119\_RBS 1](#Engineering-The-Constitutive-URS-J23119-RBS-1)
 [Engineering Process for The Keratinase KerDZ](#Engineering-Process-for-The-Keratinase-KerDZ)
 [Engineering Process for Keratin-31](#Engineering-Process-for-Keratin-31)

# Engineering The Inducible URS Series pTac\_RBS 1/2/3/4

## Design

Our first task involved creating a library of plasmids to carry the genes required for ReneWool, as well as to build on the iGEM collection of
knowledge. ReneWool includes a constitutive expression system for textile degrading enzymes, such as KerDZ, and an inducible system for spider
silk production. This combination of constitutive and inducible plasmids allows us to increase spider silk production in periods of feedstock abundance
and optimal biomass accumulation, while continuously producing the enzymes for degradation. With this in mind, we made two sets of plasmids, inducible
and constitutive, which we compared in various strains to optimize protein expression. We tested different upstream regulatory sites in two
plasmids from the pJUMP series and gathered data on protein expression and compatibility with our selected strains.

Our main experimental vectors were pJUMP24-1A, to carry the inducible promoters, and pJUMP28-1A, to carry the constitutive promoters. Apart
from their origins of replication, these plasmids are similar in structure, allowing data gathered from one characterization experiment to be
extrapolated to the other. They are also compatible with each other, owing to their different origins of replication, which allows us to design an
expression system within a single organism capable of degrading textiles and producing spider silk!

## Build

We began by assembling our inducible vectors. The IPTG inducible pTac promoter and one of four Ribosome Binding Sites (RBS1, RBS2, RBS3 or RBS4)1
were inserted into pJUMP24-1A via Gibson Assembly. Additionally, sfGFP was inserted downstream of the RBS as a reporter gene for expression under
each pTac\_RBS upstream regulatory site (URS). These plasmids were cloned into *Escherichia coli* strains DH5ɑ, K12, and the protein expression strains
Rosetta-gami™(Hereby referred to as Rosetta-gami) and BL21. To select for isolated colonies containing
our desired plasmid, the transformed strains were grown on LB agar containing 50 µg/ml Kanamycin.

![](https://static.igem.wiki/teams/5427/pictures-of-constructs/rbs1-2-3-4-sfgfp-pjump24.webp)

**Fig 1. |**
Our pJUMP24-1A\_pTac\_RBS(1-4)\_sfGFP series of plasmids.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Test

We carried out several characterization experiments to determine the optimal growth conditions for transformants carrying the
plasmid, the metabolic burden of our inserts, and the strength of each pTac\_RBS upstream regulatory site.

First, we conducted growth experiments on *E. coli* DH5ɑ, K12, Rosetta-gami, and BL21 strains transformed with
pJUMP24-1A\_pTac\_RBS(1-4)\_sfGFP. This was done to determine the optimal temperature for growth of transformed cultures.
Additionally, we tested the metabolic burden of pTac\_RBS(1-4)\_sfGFP
on each strain by comparing the biomass accumulation of our clones with that of the empty cell controls.

![](https://static.igem.wiki/teams/5427/pictures-for-parts/ptac-rbs1-2-3-4-sfgfp-pjump24-dh5a-bl21-k12-rosetta-gami-at-30.webp)

**Fig 2. |**
Growth curves of DH5ɑ, BL21, K12 and Rosetta-gami strains carrying pJUMP24-1A\_pTac\_RBS(1-4)\_sfGFP. Empty cell controls
for each strain were grown in LB, and transformants were grown in LB Kanamycin (50µg/ml). All cultures were grown at 30oC
for ten hours. The Optical Density
(OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

![](https://static.igem.wiki/teams/5427/pictures-for-parts/ptac-rbs1-2-3-4-sfgfp-pjump24-dh5a-bl21-k12-rosetta-gami-at-37.webp)

**Fig 3. |**
Growth curves of DH5ɑ, BL21, K12, and Rosetta-gami strains carrying pJUMP24-1A\_pTac\_RBS(1-4)\_sfGFP. Empty cell controls for each
strain were grown in LB, and transformants were grown in LB Kanamycin (50µg/ml). All cultures were grown
at 37oC for ten hours. The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

The growth curves demonstrated that 37oC is optimal for the propagation of pJUMP24-1A\_pTac\_RBS\_sfGFP, as cultures at this temperature
obtained higher biomass accumulation compared to cultures at 30oC. Although visually there appears to be variation in growth rate between
transformants, no statistically significant difference was observed between any of the RBSs or between the RBSs and the empty cell control.
From this data, we concluded that pTac\_RBS1, 2, 3, and 4 do not impose a considerable metabolic burden on any of the strains tested.
However, we still wanted to determine the relative strength of each RBS.

## Test

In tandem with the above growth curve experiment, we tested the degree of protein expression under the pTac\_RBS upstream regulatory sites.
Our team conducted a fluorometric characterization experiment. We induced sfGFP expression in *E. coli* DH5ɑ carrying pJUMP24-1A\_pTac\_RBS1/2/3/4\_sfGFP with
IPTG, lysed the cells via French Press, and measured the fluorescence of the pellet after lysis.

![](https://static.igem.wiki/teams/5427/contribution-assets/contribution-figure-5-eng-sucess-figure-4-exp-results-figure-7-fluorescence-data-for-sfgfp-isolated-from-dh5a-conatning-the-ptac-rbs-1-2-3-4-sfgfp-pjump-24-plasmid-after-3-hour-induction-with-3-mm-iptg.webp)

**Fig 4. |**
Fluorescence of *E. coli* DH5ɑ cultures transformed with pJUMP24-1A\_pTaq\_RBS(1-4)\_sfGFP and induced with 3mM IPTG in liquid LB Kanamycin
(50µg/ml). The reported fluorescence values have been background (fluorescence of empty DH5ɑ cells)
subtracted to account for non-specific signals.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

RBS1 transformants reported the strongest fluorescence, indicating that pTac\_RBS1 is the strongest URS of those tested. As such, pTac\_RBS1 was selected as the URS for our
spider silk gene, which needs to be inducible to maximize silk production. However, the fluorescence observed in RBS1 transformants was lower than is generally expected for
sfGFP, implying that
although RBS1 was the stronger RBS of our series, pJUMP24-1A\_pTac\_RBS1 is not an overly strong expression vector.

# Engineering Process for Spider Silk

## Design

Our spider silk gene, (A3I)3-A14, encodes a modified version of the recombinant spider silk NT2RepCT.2 We selected this protein due to its incredible physical properties,
which match those of native dragline spider silk.3 We optimized this spider silk gene for expression in *E. coli* using the IDT Codon Optimization Tool,
attached a 6xHis tag to the N-terminus, and added the standard BioBrick prefix and suffix around the gene.

## Build

As pTac\_RBS1 was the strongest inducible URS tested in our pJUMP24-1A\_pTac\_RBS1/2/3/4\_sfGFP characterizations, it was selected as the URS for the expression of our spider silk gene. sfGFP was added in tandem
to spider silk to create a translational fusion that allows for detection of spider silk production.

![](https://static.igem.wiki/teams/5427/pictures-of-constructs/rbs1-spidersilk-sfgfp-pjump24.webp)

**Fig 5. |**
Our pJUMP24-1A\_pTac\_RBS1\_SpiderSilk\_sfGFP plasmid.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Test

To test whether our spider silk gene imposes a significant metabolic burden on the cell—which would impact the feasibility of producing
spider silk on a
commercial scale—we tested the growth of DH5ɑ transformed with pJUMP24-1A\_pTac\_RBS1\_SpiderSilk\_sfGFP.

![](https://static.igem.wiki/teams/5427/experiments-and-results-assets/growth-curve-for-ptac-rbs-1-spider-silk-sfgfp-pjump-24-in-dh5a-at-37-degrees-in-regular-lb.png)

**Fig 6. |**
Growth curve of DH5ɑ transformed with pJUMP24-1A\_pTac\_RBS1\_SpiderSilk\_sfGFP. Empty cell controls for each strain were grown in LB, and
transformants were grown in LB Kanamycin (50µg/ml). Cultures were grown at 37oC for ten hours.
The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

DH5ɑ pJUMP24-1A\_pTac\_RBS1\_SpiderSilk\_sfGFP transformants grew more slowly and reached lower overall biomass than empty DH5ɑ cells.
This result indicated that the spider silk containing plasmid imposes a metabolic burden on cells. This may pose a problem for using this
expression vector to produce spider silk on a
commercial scale. See
[future directions

Future Directions

![](https://static.igem.wiki/teams/5427/screenshots/future-screenshot.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27future%27%29%20%7D%7D)
for our proposed new designs and considerations.

# Engineering The Constitutive URS J23119\_RBS 1

## Design

In addition to the inducible URS described above, the ReneWool system requires a constitutive expression system for textile
degrading enzymes. In designing our constitutive system, we applied what
we learned from the experiments outlined above, as well as research from the 2023 UAlberta iGEM team.
The 2023 UAlberta iGEM team had success using the J23 series of promoters in pJUMP vectors.4 To expand on their knowledge, we chose to
experiment with the constitutive promoter J23119 in pJUMP28-1A. pJUMP28-1A was chosen due to its similarity to pJUMP24-1A and their ability
to be expressed together in a single organism. We chose RBS1 as the ribosomal binding site, as
our previous results indicated this was the strongest RBS in our series

![](https://static.igem.wiki/teams/5427/pictures-of-constructs/j23119-rbs1-pjump28.webp)

**Fig 7. |**
Our constitutive pJUMP28-1A\_J23119\_RBS1 plasmid. This constitutive expression system is necessary for our textile-degrading enzymes.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Build-Test

Following BioBrick standards, we cloned J23119\_RBS1 into pJUMP28-1A via restriction cloning using EcoRI and PstI cut sites in the biobrick
sites on both the pJUMP 28-1A vector and the J23119\_RBS 1 URS. To test the metabolic burden of J23119\_RBS1, we transformed DH5ɑ, Rosetta-gami,
and BL21 with pJUMP28-1A\_J23119\_RBS1 and performed a growth experiment.

![](https://static.igem.wiki/teams/5427/contribution-assets/growth-curve-for-j23119-rbs-1-pjump-28-in-dh5a-r-gami-and-bl21-at-37-degrees-in-regular-lb.webp)

**Fig 8. |**
Growth curve of DH5ɑ, BL21, and Rosetta-gami strains carrying pJUMP28-1A\_J23119\_RBS1. Empty cell controls for each strain were grown in LB,
and transformants were grown in LB Kanamycin (50µg/ml). All cultures were grown at 37oC for 10 hours.
The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

DH5ɑ transformants achieved the highest OD600 out of the tested strains, with Rosetta-gami transformants achieving the next highest. Interestingly,
although BL21 transformants achieved the lowest biomass accumulation of the three transformed strains,
transformed BL21 attained a greater OD than empty BL21
Taken together, these results suggest that J23119\_RBS1 does not impose a large metabolic burden and that DH5ɑ is superior to BL21 and
Rosetta-gami in terms of plasmid maintenance. Nevertheless, we still wanted to test whether DH5ɑ remained the ideal strain
once kerDZ was incorporated into the plasmid, as DH5ɑ is not a protein expression strain.

## Test

As Kanamycin is susceptible to high salt concentrations, we also conducted this experiment in Lennox LB (43.1 mM NaCl) and regular LB
(86.2 mM NaCl) to determine
the optimal growing conditions for maintaining pJUMP28-1A\_J23119\_RBS1 in DH5ɑ transformants.

![](https://static.igem.wiki/teams/5427/experiments-and-results-assets/j23119-dh5a-low-and-normal-salt.png)

**Fig 9. |**
Growth curve of DH5ɑ carrying pJUMP28-1A\_J23119\_RBS1 grown in LB (86.2 mM NaCl) Kanamycin (50µg/ml) or Lennox LB (43.1 mM NaCl)
Kanamycin (50µg/ml). Cultures were grown at 37oC over 6 hours.
The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

We observed no significant difference between DH5ɑ growth at high and low salt concentrations. If a higher rate of cell growth were achieved in the higher salt
media, that would imply Kanamycin was being deactivated by the salt, causing our plasmids to be lost and cells to grow faster as a result. Based on our data, Kanamycin
is not affected by salt concentration,
and either condition is likely suitable for maintaining the pJUMP28\_J23119\_RBS1 plasmid in DH5ɑ, Rosetta-gami, or BL21 strains.
The data gathered on the constitutive pJUMP28-1A\_J23119\_RBS1 plasmid informed the design of our KerDZ part. As pJUMP28\_J23119\_RBS1 did
not impose a large metabolic burden on cells, it was selected as the plasmid for KerDZ.

# Engineering Process for The Keratinase KerDZ

## Design

To design our keratin-degrading enzyme, we searched the literature for a keratinase that would function efficiently in our system. KerDZ was selected based on
its relatively high thermostability, ability to function at a wide range of pHs, and its known ability to degrade wool when expressed in *E. coli*.5, 6 We optimized kerDZ,
originally from *Actinomadura viridilutea*, for expression in *E. coli* using the IDT Codon Optimization tool,
attached a 6xHis tag to the N-terminus, and added the standard BioBrick prefix and suffix around the gene.

## Build

Following BioBrick standards, we cloned kerDZ, the constitutive promoter J23119, and RBS1 into pJUMP28-1A via restriction cloning using the SpeI and PstI cut
sites for the J23119\_RBS 1\_pJUMP 28 vector and the XbaI and PstI cut sites for the KerDZ gene fragment. We transformed DH5ɑ with this plasmid for propagation
and later transformed it into BL21 and Rosetta-gami to achieve constitutive expression of KerDZ in our system.

![](https://static.igem.wiki/teams/5427/pictures-of-constructs/kerdz.webp)

**Fig 10. |**
Our constitutive pJUMP28-1A\_J23119\_RBS1\_KerDZ plasmid.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

This construct incorporated data from the fluorometric characterization of pJUMP24-1A\_pTac\_RBS1/2/3/4\_sfGFP, which revealed that RBS1 was
the strongest of our RBS series. Additionally, the pJUMP28-1A\_J23119\_RBS1 growth curve (**Figure 8**) confirmed
that J23119\_RBS1 does not impose a significant metabolic burden on DH5ɑ, Rosetta-gami, or BL21.

## Test

To test the metabolic burden of J23119\_RBS1\_KerDZ, we transformed DH5ɑ, Rosetta-gami,
and BL21 with pJUMP28-1A\_J23119\_RBS1\_KerDZ and performed the following growth experiments.

![](https://static.igem.wiki/teams/5427/engineering-success-assets/growth-curve-for-j23119-rbs-1-kerdz-pjump-28-in-dh5a-r-gami-and-bl21-at-37-degrees-in-lennox-lb.png)

**Fig 11a. |**
Growth curve of DH5ɑ, BL21, and Rosetta-gami strains carrying pJUMP28-1A\_J23119\_RBS1\_KerDZ in Lennox LB (43.1 mM NaCl).
Empty cell controls for each strain were grown in LB, and transformants were grown in LB Kanamycin (50µg/ml).
Cultures were grown at 37oC for 10 hours. The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

![](https://static.igem.wiki/teams/5427/experiments-and-results-assets/growth-curve-for-j23119-rbs-1-kerdz-pjump-28-in-dh5a-r-gami-and-bl21-at-37-degrees-in-regular-lb.png)

**Fig 11b. |**
Growth curve of DH5ɑ, BL21, and Rosetta-gami strains carrying pJUMP28-1A\_J23119\_RBS1\_KerDZ in LB (86.2 mM NaCl). Empty cell controls
for each strain were grown in LB, and transformants were grown in LB Kanamycin (50µg/ml). Cultures were grown at 37oC
for 10 hours. The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

We observed that DH5ɑ and BL21 transformants achieved higher biomass accumulation than Rosetta-gami transformants, indicating
that these strains are optimal for pJUMP28-1A\_J23119\_RBS1\_KerDZ plasmid propagation. Once again, BL21 transformants grew faster
and achieved a higher overall biomass than empty BL21 cells, emphasizing that the pJUMP28-1A\_J23119\_RBS1\_KerDZ plasmid does not impose
a significant metabolic burden on BL21 cells. Although DH5ɑ transformants did not reach the same optical density as empty DH5ɑ cells,
this difference is not significant, implying
that either of these strains would be suitable for pJUMP28-1A\_J23119\_RBS1\_KerDZ plasmid propagation.
At a glance, transformant growth in Lennox LB (**Figure 22a**) appears slower, and transformants in this media reached lower overall biomass
compared to those in regular LB. While there may be a link between salt concentration and the activity of Kanamycin, this relationship remains
unclear as there is not a
statistically significant difference in growth rate.

# Engineering Process for Keratin-31

![](https://static.igem.wiki/teams/5427/modeling-assets/fig7a-kerdz-keratin31-interaction.webp)

**Fig 12. |**
Model of Keratin-31 interaction with KerDZ. Created using
[HDOCK
HDOCK

![](https://static.igem.wiki/teams/5427/screenshots/hdock-screenshot.webp)](http://hdock.phys.hust.edu.cn/)
by inputting
[AlphaFold2
AlphaFold2

![](https://static.igem.wiki/teams/5427/screenshots/alphafold-screenshot.webp)](https://alphafold.ebi.ac.uk/)
models of Keratin-31 and KerDZ.
The interaction was then visualized with
[SWISS-MODEL
SWISS-MODEL

![](https://static.igem.wiki/teams/5427/screenshots/swiss-screenshot.webp)](https://swissmodel.expasy.org/).

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

To test the efficiency of our keratinolytic enzyme, we aimed to express keratin in our system.
*krt31* was optimized for expression in *E. coli* using the IDT Codon Optimization tool. We
attached a 6xHis tag to the C-terminus to allow for downstream Immunoaffinity Chromatography (IMAC) and added the standard BioBrick
prefix and suffix around the gene. In parallel to the above experiments, we used Gibson Assembly to clone *krt31* downstream of the inducible
T7 promoter in the pET22-b(+) backbone. This design was chosen because of the reliability of the pET vector and the T7 promoter. Additionally,
pET22-b(+) was an ideal vector because it is
compatible with the pJUMP series and contains a pelB sequence, which aids in protein purification.

![](https://static.igem.wiki/teams/5427/pictures-of-constructs/keratin.webp)

**Fig 13. |**
Our inducible pET22-b(+)\_T7\_Kert31 plasmid.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Test

To test whether the keratin-containing plasmid imposes a significant metabolic burden on the cell,
we performed growth experiments on DH5ɑ, Rosetta-gami and BL21 transformed with pET22-b(+)\_T7\_*krt31*.

![](https://static.igem.wiki/teams/5427/experiments-and-results-assets/growth-curve-keratin-31-pet22b-in-dh5a-r-gami-and-bl21-at-37-degrees-in-regular-lb.png)

**Fig 14. |**
Growth curves of DH5ɑ, BL21, and Rosetta-gami strains carrying pET22-b(+)\_T7\_*krt31*. Empty cell controls for each strain were grown in LB, and
transformants were grown in LB Ampicillin (100µg/ml). Cultures were
grown at 37oC for 10 hours. The Optical Density (OD) at 600nm of each culture was measured every two hours.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

We observed that DH5ɑ transformants achieved the highest biomass accumulation, followed by Rosetta-gami transformants.
This result indicates that these strains are optimal for pET22-b(+)\_T7\_*krt31* plasmid propagation. Although BL21 transformants
grew slower than DH5ɑ and Rosetta-gami transformants, their growth is very similar to that of empty BL21 cells. Combined with the
relatively high biomass achieved by Rosetta-gami and DH5ɑ transformants, this implies that pET22-b(+)\_T7\_*krt31* does not impose a significant
metabolic burden on these strains and that they are suitable for further experimentation.

## Test

To test for Krt31 expression in this vector we transformed pET22-b(+)\_T7\_*krt31* into the *E. coli* Rosetta-gami and BL21
protein expression strains. The plasmid was purified and sequenced and was confirmed to contain Krt31 in the correct frame.
To characterize protein expression, we induced the strains with 1 mM IPTG overnight and compared the protein levels with uninduced
strains. After induction, we lysed the cells using either French Press or Bacterial-Protein Extraction Reagent (B-PER) and collected
the lysate for further purification. We attempted to purify Krt31 from the lysate using Immobilized Metal Affinity Chromatography
(IMAC). To verify for successfully purified keratin, we
ran the purified lysates on 12% SDS-PAGE gels and visualized using silver staining

## Learn

Krt31, which is approximately 46kDa, did not appear on our silver-stained gel. In response, we did further research on
Krt31 and discovered it is highly hydrophobic and is unlikely to be found in the soluble lysate. Instead, it was likely
to be left in the pellet after lysis. Additionally, the low amount of protein visualized on
our silver-stained gel led us to western blotting, which is more sensitive and specific.

## Test

We set up the next protein purification in a similar manner. However, after lysis using French Press or B-PER, we collected the
remaining pellet. We again purified the soluble lysate using IMAC but ran both the resuspended pellet and the lysate on a 12% SDS-PAGE gel.
We then visualized the soluble and insoluble fractions
using Western blotting with a MouseɑHis primary antibody and a GoatɑMouse secondary antibody.

## Learn

No 46kDa bands appeared in either the Rosetta-gami or BL21 induced and uninduced pellets. However, two bands approximately 36 and
34 kDa appeared in all Rosetta-gami and BL21 strains, including the untransformed controls. This is indicative of either contamination with a 6xHis
tagged protein, or the non-specific binding of our antibodies to proteins native to both Rosetta-gami and BL21. Additionally, the presence of a faint
band around 20kDa in the Rosetta-gami strain induced with 1mM IPTG lane likely suggests non-specific antibody binding to a protein unique to Rosetta-gami,
and potentially inducible by IPTG. In any case, the numerous bands that appeared on this blot indicated that 6xHis antibodies were not specific enough to
Krt31. The absence of any band corresponding to Krt31 indicates that our Krt31 was not purified from either the lysate or the pellet. Further research
into the purification of insoluble proteins yielded a new protocol for the isolation of insoluble proteins from inclusion bodies; this was likely where
Krt31 was
stored, which explains why our antibodies were not capable of binding its 6xHis tag.

## Test

Unsuccessful attempts at purifying the expressed Krt31 protein led us to design a new protocol for the purification of Krt31. A key
change to the previous protocols included the addition of a modified solubilization buffer [25 mM Tris-HCl, 9.5 M Urea, 5 mM DTT, 2.5 mM EDTA]
which was previously shown to be effective in the purification of Keratin 35 and 857 in a 1:1.5 ratio to the pelleted cells. Additionally,
rather than attempting cell lysis with French Press or B-PER, we used sonication to lyse the cells by giving them 10-second shocks, followed by
10-second rests, for thirteen cycles. From here, we
performed IMAC with modified buffers to optimally and selectively purify our protein.

![](https://static.igem.wiki/teams/5427/engineering-success-assets/image.webp)

**Fig 15. |**
Coomassie-stained 12% SDS PAGE gel showing Krt31 and KerDZ from soluble and insoluble fractions of sonicated
Rosetta-gami and BL21. The signal in the first lane at approximately 36 kDa may include Krt31.

![](https://static.igem.wiki/teams/5427/icons/x.svg)

## Learn

After several attempts at redesigning our Krt31 purification protocol we finally began to see progress. The protein purified
from the induced Rosetta-gami pellet showed a strong signal at roughly 36 kDa, slightly smaller than the expected size of Krt31.
Still, that the strong expression was found only in the induced strains implies that this may be the isolated keratin. While this is not a
definitive result, it clearly shows progress in the right direction. Future studies would involve further refinement of this protocol and could
utilize RT-PCR to
uncover the exact nature of *krt31* expression. See
[future directions

Future Directions

![](https://static.igem.wiki/teams/5427/screenshots/future-screenshot.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27future%27%29%20%7D%7D)
for more information.

References

1. Ryu, M.-H., and Gomelsky, M. (2014) Near-infrared light responsive synthetic C-di-GMP module for Optogenetic Applications.
   *ACS Synthetic Biology 3, 802–810.*
2. Andersson M, Jia Q, Abella A, Lee X-Y, Landreh M, Purhonen P, Hebert H, Tenje M, Robinson CV, Meng Q, et al. **2017**. Biomimetic
   spinning of artificial spider silk from a chimeric minispidroin. Nat Chem Biol. 13(3):262–264. doi:
   <https://doi.org/10.1038/nchembio.2269>
3. Arndt T, Greco G, Schmuck B, Bunz J, Shilkova O, Francis J, Pugno NM, Jaudzems K, Barth A, Johansson J, et al. **2022**. Engineered
   Spider Silk Proteins for Biomimetic Spinning of Fibers with
   Toughness Equal to Dragline Silks. Adv Funct Materials. 32(23):2200986. doi:
   <https://doi.org/10.1002/adfm.202200986>
4. UAlberta iGEM. 2023. Parts | UAlberta - iGEM 2023.
   <https://2023.igem.wiki/ualberta/parts>
   (accessed 2024-09-24).
5. Ben Elhoul M, Zaraî Jaouadi N, Rekik H, Omrane Benmrad M, Mechri S, Moujehed E, Kourdali S, El Hattab M, Badis A, Bejar S,
   et al. **2016**. Biochemical and molecular characterization of new keratinoytic
   protease from *Actinomadura viridilutea* DZ50. Int. J. Biol. Macromol.. 92:299–315. doi:
   <https://doi.org/10.1016/j.ijbiomac.2016.07.009>
6. Ben Elhoul M, Zaraî Jaouadi N, Bouacem K, Allala F, Rekik H, Mechri S, Khemir Ezzine H, Miled N, Jaouadi B. **2021**. Heterologous
   expression and
   purification of keratinase from *Actinomadura viridilutea* DZ50: feather biodegradation and animal
   hide dehairing bioprocesses. Environ Sci Pollut Res. 28(8):9921–9934. doi:
   <https://doi.org/10.1007/s11356-020-11371-1>
7. Ishii, D., Abe, R., Watanabe, S., Tsuchiya, M., Nöcker, B., and Tsumoto, K. (2011) Stepwise characterization of the thermodynamics of trichocyte
   intermediate filament protein supramolecular assembly. *Journal of Molecular Biology* 408, 832–838.

{% endblock %}