iGEM 2021 — Team IISc-Bangalore

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# Team:IISc-Bangalore/Notebook

Lab Notebook | IISc Bangalore

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* [Judging](https://2021.igem.org/Team%3AIISc-Bangalore/Judging)

* [Description](https://2021.igem.org/Team%3AIISc-Bangalore/Description)
* [Design](https://2021.igem.org/Team%3AIISc-Bangalore/Design)
* [Engineering](https://2021.igem.org/Team%3AIISc-Bangalore/Engineering)
* [Implementation](https://2021.igem.org/Team%3AIISc-Bangalore/Implementation)
* [Proof of Concept](https://2021.igem.org/Team%3AIISc-Bangalore/Proof_Of_Concept)
* [UN SDG](https://2021.igem.org/Team%3AIISc-Bangalore/Sustainable)
* [Safety](https://2021.igem.org/Team%3AIISc-Bangalore/Safety)
* [Contribution](https://2021.igem.org/Team%3AIISc-Bangalore/Contribution)

* [Results](https://2021.igem.org/Team%3AIISc-Bangalore/Results)
* [Lab Notebook](https://2021.igem.org/Team%3AIISc-Bangalore/Notebook)

* [Model](https://2021.igem.org/Team%3AIISc-Bangalore/Model)
* [Protein Structure
  Predictions](https://2021.igem.org/Team%3AIISc-Bangalore/Predictions)

* [Education and
  Communication](https://2021.igem.org/Team%3AIISc-Bangalore/Communication)
* [Integrated Human
  Practices](https://2021.igem.org/Team%3AIISc-Bangalore/Human_Practices)

* [Members](https://2021.igem.org/Team%3AIISc-Bangalore/Members)
* [Attributions](https://2021.igem.org/Team%3AIISc-Bangalore/Attributions)
* [Collaborations](https://2021.igem.org/Team%3AIISc-Bangalore/Collaborations)

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   2. [Lab notebook](https://2021.igem.org/Team%3AIISc-Bangalore/Notebook)
5. Drylab
   1. [Model](https://2021.igem.org/Team%3AIISc-Bangalore/Model)
   2. [Protein Structure Predictions](https://2021.igem.org/Team%3AIISc-Bangalore/Predictions)
6. Human Practices:
   1. [Education
      and Communication](https://2021.igem.org/Team%3AIISc-Bangalore/Communication)
   2. [Integrated Human
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7. Team:
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   3. [Collaborations](https://2021.igem.org/Team%3AIISc-Bangalore/Collaborations)
8. [Judging](https://2021.igem.org/Team%3AIISc-Bangalore/Judging)

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# Lab Notebook

Like most synthetic biology projects, wet lab work was an integral part of our project. We worked in
multiple laboratories for different parts of our projects - protein production and Bacterial
Cellulose or Microcrystalline Cellulose Sheet Production - for different durations. The lab notebook
provided here is a documentation of all the work we did in these laboratories.

The entire lab notebook for our project is attached below as a PDF file. We have combined the work
done in the 4 different labs during the entire duration.

The first 2 pages contain the buffers and the buffers' compositions that were used during our
experiments, mostly in protein production.

The order of the labs in which the work has been presented is as follows:

1. **Dr. Palani's Lab**: - Protein Production and Purification
2. **Prof. Ramamurthy's Lab**: - Making Microcrystalline Cellulose (MCC) Sheets from MCC powder
3. **Prof. Chakravortty's Lab and Dr. Pande's Lab**: - Production of Bacterial Cellulose from
   *Agrobacteriumtum faciens* and *Komagateibacter xylinus*

We have presented all the experiments in a day-wise arrangement. The results have been shown after
the corresponding experiments. We have mentioned protocols for basic molecular biology laboratory
techniques and have summarised all relevant details about our results and conclusions. All relevant
gel images have also been inserted appropriately. The lab work has been labelled by the date of the
experiment and the lab in which it has been carried out. We believe that this extensive
documentation of our wetlab work will be of importance to future iGEM teams who choose to work in
this field.

[Download Lab
Notebook](https://static.igem.org/mediawiki/2021/d/dd/T--IISc-Bangalore--pdfs--Lab_notebook_final.pdf)

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# Team:IISc-Bangalore/Engineering

Engineering | IISc Bangalore

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* [Judging](https://2021.igem.org/Team%3AIISc-Bangalore/Judging)

* [Description](https://2021.igem.org/Team%3AIISc-Bangalore/Description)
* [Design](https://2021.igem.org/Team%3AIISc-Bangalore/Design)
* [Engineering](https://2021.igem.org/Team%3AIISc-Bangalore/Engineering)
* [Implementation](https://2021.igem.org/Team%3AIISc-Bangalore/Implementation)
* [Proof of Concept](https://2021.igem.org/Team%3AIISc-Bangalore/Proof_Of_Concept)
* [UN SDG](https://2021.igem.org/Team%3AIISc-Bangalore/Sustainable)
* [Safety](https://2021.igem.org/Team%3AIISc-Bangalore/Safety)
* [Contribution](https://2021.igem.org/Team%3AIISc-Bangalore/Contribution)

* [Results](https://2021.igem.org/Team%3AIISc-Bangalore/Results)
* [Lab Notebook](https://2021.igem.org/Team%3AIISc-Bangalore/Notebook)

* [Model](https://2021.igem.org/Team%3AIISc-Bangalore/Model)
* [Protein Structure
  Predictions](https://2021.igem.org/Team%3AIISc-Bangalore/Predictions)

* [Education and
  Communication](https://2021.igem.org/Team%3AIISc-Bangalore/Communication)
* [Integrated Human
  Practices](https://2021.igem.org/Team%3AIISc-Bangalore/Human_Practices)

* [Members](https://2021.igem.org/Team%3AIISc-Bangalore/Members)
* [Attributions](https://2021.igem.org/Team%3AIISc-Bangalore/Attributions)
* [Collaborations](https://2021.igem.org/Team%3AIISc-Bangalore/Collaborations)

Navigation dropdown

1. [Home](https://2021.igem.org/Team%3AIISc-Bangalore)
2. Project:
   1. [Description](https://2021.igem.org/Team%3AIISc-Bangalore/Description)
   2. [Design](https://2021.igem.org/Team%3AIISc-Bangalore/Design)
   3. [Engineering](https://2021.igem.org/Team%3AIISc-Bangalore/Engineering)
   4. [Implementation](https://2021.igem.org/Team%3AIISc-Bangalore/Implementation)
   5. [Proof of Concept](https://2021.igem.org/Team%3AIISc-Bangalore/Proof_Of_Concept)
   6. [UN SDG](https://2021.igem.org/Team%3AIISc-Bangalore/Sustainable)
   7. [Safety](https://2021.igem.org/Team%3AIISc-Bangalore/Safety)
   8. [Contribution](https://2021.igem.org/Team%3AIISc-Bangalore/Contribution)
3. [Parts](https://2021.igem.org/Team%3AIISc-Bangalore/Parts)
4. Lab work:
   1. [Results](https://2021.igem.org/Team%3AIISc-Bangalore/Results)
   2. [Lab notebook](https://2021.igem.org/Team%3AIISc-Bangalore/Notebook)
5. Drylab
   1. [Model](https://2021.igem.org/Team%3AIISc-Bangalore/Model)
   2. [Protein Structure Predictions](https://2021.igem.org/Team%3AIISc-Bangalore/Predictions)
6. Human Practices:
   1. [Education
      and Communication](https://2021.igem.org/Team%3AIISc-Bangalore/Communication)
   2. [Integrated Human
      Practices](https://2021.igem.org/Team%3AIISc-Bangalore/Human_Practices)
7. Team:
   1. [Members](https://2021.igem.org/Team%3AIISc-Bangalore/Members)
   2. [Attributions](https://2021.igem.org/Team%3AIISc-Bangalore/Attributions)
   3. [Collaborations](https://2021.igem.org/Team%3AIISc-Bangalore/Collaborations)
8. [Judging](https://2021.igem.org/Team%3AIISc-Bangalore/Judging)

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# Engineering

In this page, we will introduce the Engineering Design Cycle that we followed in order to eventually
accomplish our short-term goals. We followed this workflow for all four components that constitute
CellOPHane: protein production, bacterial cellulose (BC) production, MCC sheet production, and
hardware development. During this process, successful results were obtained, but also new challenges
were discovered. Nevertheless, the drawbacks forced us to come up with new ideas and initiated new
cycles of engineering, ultimately driving our project forward. While the exact experiments and
results are described on the Results page, here we outline the underlying thought process that led
to our final engineering success.

Contents:

* [Proteins:](#proteins)

1. [Cycle 1](#p1)
2. [Cycle 2](#p2)
3. [Cycle 3](#p3)
4. [Cycle 4](#p4)

* [Bacterial Cellulose Production:](#bacterial)

1. [Cycle 1](#b1)
2. [Cycle 2](#b2)
3. [Cycle 3](#b3)
4. [Cycle 4](#b4)

* [MCC Sheet:](#mcc)

1. [Cycle 1](#m1)
2. [Cycle 2](#m2)
3. [Cycle 3](#m3)

* [Hardware:](#hardware)

1. [Cycle 1](#h1)

* [References](#ref)

Synthetic biology is a discipline of science that differs a lot from the others. Usually, one would
use the scientific method to obtain results in the majority of life sciences, centering one 's work
on making observations and performing experiments. However, SynBio goes beyond that! It is all about
constructing something new from parts (biological parts if you may). That is why, sometimes the
engineering design process fits better with these projects, since a product has to be built or a
"genetically engineered machine" has to perform a specific task. It reminds us that iGEM stands for
International Genetically Engineered Machines.

![](https://static.igem.org/mediawiki/2021/a/ae/T--IISc-Bangalore--images--engineering--Engg.png)

## Proteins

### Cycle 1

Design

After long search through literature, we selected dCBD and OpdA. The former was selected
for its strong, synergistic binding with microcrystalline cellulose. The latter was
selected for its broad substrate range that includes a couple of extremely hazardous
pesticides which are heavily used in Indian agricultural fields. We chose sfGFP as the
perfect reporter for our purpose.

Initially we tried to incorporate SpyDock in fusion protein C1 and SpyTag002 in fusion
protein C2. Maintaining modularity was our first and foremost priority while designing
the filter platform, and we planned to utilize noncovalent interaction in binding C1
with C2. SpyDock is a modified version of SpyCatcher which binds in 1:1 stoichiometry
with SpyTag002 via noncovalent interaction, and not irreversible isopeptide linkage.
SpyTag/SpyDock system is at the heart of Spy&Go resin that is used for protein
purification. We planned to functionalize the bacterial cellulose (BC) sheet by
incubating it with cocktail of C1 (SpyDock-dCBD) and C2 (OpdA-SpyTag002-sfGFP). After
the functionalized filter gets installed (as a part of the associated hardware), the BC
base of the filter would eventually get worn out with time. Thereafter, we can just flow
2.5M imidazole solution through our filter. It would elute out the C2 molecules from the
filter, which can be purified and reused later. Thus, adopting this strategy would
enable one of our two fusion proteins to be reusable. Most importantly, C2 being larger
than C1 we hoped that this strategy would make our filtration process more economically
viable.

Build

Before stepping into wet lab work, we designed a first-order mathematical model on
SpyTag/SpyDock interaction. We searched for parametric values required to run this model
and fed those to it.

Test

Upon running computer simulations, the model yielded plots regarding the extent of
SpyTag/SpyDock complex dissociation at various volumetric flow rates.

Learn

The model showed that SpyTag/SpyDock interaction gets abolished after a short period of
time and, as a result, the process of C2 getting washed away has a very low lifetime.
Though we hoped that the noncovalent interaction-based strategy would decrease the cost
as well as would make C2 reusable, we learnt from the model this strategy would take us
nowhere. Thus, the model safely ruled out the incorporation of SpyTag/SpyDock into the
constructs we would design next.

### Cycle 2

Design

The previous cycle made our focus shift to covalent protein ligation strategies instead.
The SpyTag/SpyCatcher system readily attracted our attention. The interaction between
the two is covalent, which means the binding is essentially irreversible. The reaction
requires only mixing and is rapid, high yielding, robust to experimental conditions, and
shows very good specificity. The system is functional over a wide range of temperature,
pH and salinity. SpyTag002 and SpyCatcher002 were the preferred versions. We planned to
incorporate SpyCatcher002 in fusion protein C1 and SpyTag002 in fusion protein C2. We
also thought of some back-up plans. We included KpnI restriction sites on either end of
the sfGFP CDS, should the need of removing sfGFP ever arises.

Build

We designed two constructs - C1 (SpyCatcher002-dCBD) and C2 (OpdA-SpyTag002-sfGFP). To
know the efficiency of our system, both C1 and C2 proteins had to be synthesized, which
was carried out using E. coli DH5 alpha as the cloning host and *E.
coli* BL21(DE3) as the
expression host. The specific genes for dCBD, OpdA, and sfGFP were derived from *T.
reesei, A. radiobacter, and A. victoria* respectively. We codon optimized the
genes for
expression in *E. coli* with SnapGene 's codon optimizing tool. pETMCN-T7 (KanR)
was the
vector we intended to use in our experiments and it included features (e.g. T7 promoter,
lac operator, and ribosomal binding site) that allowed for the inducible gene
expression. Additionally, both constructs have a 10x-His tag for subsequent protein
purification. We ordered the DNA sequences of both C1 and C2 from GenScript. They were
shipped to us after being cloned into pUC57-Kan vectors.

Test

After amplifying the constructs using suitable primers, we cloned each of them into
pETMCN-T7 (KanR) using Gibson assembly. It yielded C1\_pETMCN-T7 and C2\_pETMCN-T7 which
were subsequently transformed into *E. coli* DH5 alpha. Inoculation into liquid
culture
was
followed by miniprepping. Plasmid obtained from positive clones (as per double digestion
verification) was transformed into *E. coli* BL21(DE3) and secondary liquid
cultures were
induced with different working concentration of IPTG and under a range of temperatures.

SDS-PAGE of lysed cell pellets, conducted as the expression test, was inconclusive.
Thus, we performed a western blot using anti-His primary antibody. The results showed
prominent bands at ~25 kDa for C1 and at ~75 kDa for C2. It helped us to finalize the
optimal induction conditions (namely 0.5 mM IPTG at 30°C) as well.

To perform solubility test, first we followed these conditions to induce a large scale
*E. coli* BL21(DE3) cultures. Then, harvested cells were lysed and subjected to
pulldown
with Ni-NTA. SDS-PAGE was performed on the boiled samples taken from different culture
fractions (viz., lysate, pellet, supernatant, washed and bead-bound). Bead-bound
fraction for C1 showed prominent band at (~25 kDa), but C2 did not. Next, we performed
two Western blots on the C2 pulldown samples, one using anti-His primary antibody and
the other using anti-GFP primary antibody. Later, the pulldown of C2 was performed with
an anti-GFP nanobody coupled to GST-tagged glutathione-sepharose beads and it was
followed by SDS-PAGE.

Learn

Results from the expression test tell that both C1 and C2 are getting expressed by our
expression host. No pulldown was completely successful for C2. Most probably, protein
degradation was to blame. However, results from solubility test definitely tell that C1
is soluble. C2 might not have been soluble.

### Cycle 3

Design

The lack of solubility of C2 possibly stems from undesired protein-protein interaction.
Therefore, we decided to make C2 less bulky by clipping out the sfGFP module. As
mentioned previously, we already made a provision of doing the same in the design phase
of the previous engineering cycle.

We found instances in the literature where other groups had used TB media in the
secondary culture for OpdA production.

Build

Restriction digestion of C2\_pETMCN-T7 with KpnI followed by ligation yielded
C2(2.0)\_pETMCN-T7. This was our first modified construct in quest of functional C2
protein.

Test

After verification, the plasmid was transformed into *E. coli* BL21(DE3) followed
by
Ni-NTA pulldown. We performed pulldown again using SpyDock-linked Ni-NTA bead.

We grew our expression host in Terrific Broth (TB) in addition to LB (i.e. the media
used so far).

We conducted Bradford assay (using bovine serum albumin as the reference) and OpdA
activity assay (using Coumaphos as the substrate) on the crude extract of the
large-scale secondary culture.

Learn

No prominent band at ~50 kDa, the molecular weight of C2(2.0), could be observed in
either of the pulldowns. Thus, like C2, C2(2.0) also could not be verified to be
soluble.

Enzyme production seemed to be higher in cultures grown in TB than those grown in LB.
Thus, we learnt to continue using TB as the growth medium for our protein 's expression.

The OpdA activity assay tells us that OpdA, produced by TB-grown expression host, is not
undergoing proteolytic cleavage under the expression conditions provided and is active.
This serves as an extremely important aspect of CellOPHane: its Proof of Concept

### Cycle 4

Design

The lack of solubility of C2(2.0) might stem from the hydrophobic signal peptide
attached to OpdA. Therefore, we decided to clip out the said signal peptide sequence.

Build

We performed a PCR on C2(2.0)\_pETMCN-T7 to amplify C2(2.0) minus the N-terminal signal.
This PCR fragment was cloned into an empty pETMCN-T7 vector to yield C2(3.0)\_pETMCN-T7.
This was our second modified construct in quest of functional C2 protein.

Test

As we are currently in the 'Build' phase of this engineering cycle, experiments are not
performed yet.

Learn

As we are currently in the 'Build' phase of this engineering cycle, the progress on this
is not significant enough to give us any conclusive findings.

## Bacterial Cellulose Production

### Cycle 1

Design

Making bacterial cellulose (BC) sheet is integral to our project. Initially, we planned
to achieve BC overproduction by knocking out *celG* gene in
*chvB-* Agrobacterium
tumefaciens C58 kindly provided by Prof. Ann G. Matthysse as a gift. We planned to adopt
the FLP-FRT recombination to achieve the same.

Build

Necessary primers were designed and ordered.

Test

With help of gradient PCR, the lambda Red recombinase cassette of pKD46 (AmpR) was
amplified and put under *tetR* promoter in pST-KT (KanR; TetR). Gradient PCR was
again
used to amplify the complement1-FRT-CamR-FRT-complement2 fragment present on pKD3
(CamR). Here, complement1 and complement2 denote the DNA sequence complementary to
upstream and
downstream region, respectively, of the genomic portion we intend to knock out.

The *chvB- Agrobacterium tumefaciens* C58 strain was revived from
filter paper into LB
agar plates.

Learn

The amplification of lambda Red recombinase system was successful, but that of
complement1-FRT-CamR-FRT-complement2 was not. Based on the gel electrophoresis imaging,
formation of primer dimer (PD) was probably the reason the latter failed. PD is a
potential by-product in PCR reaction where two primer molecules hybridize to each other
because of strings of complementary bases in them. The DNA polymerase amplifies the PD,
leading to competition for PCR reagents, thus potentially inhibiting amplification of
the DNA sequence targeted for PCR amplification.

No bacterial growth was found on the LB agar plates where we revived the mutant
Agrobacterium strain. As per Prof. Matthysse 's words, the root of this problem might
lie
in the cultures being very old.

### Cycle 2

Design

We realized that we do not have time to re-design the pKD3 primers from scratch or to
play with the gradient PCR conditions. Lack of mutant colony was another headache.

Thus, we gave up the knock-out strategy of ours and quickly devised an alternative. We
ordered *Komagataeibacter xylinus* from NCL, Pune. Given appropriate conditions,
it has
been reported to produce BC in excessive amount.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. BC production) of our work.

Test

The strain was inoculated from agar slant into liquid Hestrin Schramm (HS) media (pH 6).
HS hard agar (HA) plates were streaked with the same liquid culture.

Learn

No growth was seen in either liquid culture or HA plates even after sufficient time was
allowed for bacterial growth. The HS media was selected after going through available
literature on BC overproduction by *K. xylinus*. However, the instruction sheet
provided
by NCL recommended sorbitol media with pH 6.2.

### Cycle 3

Design

We planned to use sorbitol media (pH 6.2) instead of HS media.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. BC production) of our work.

Test

The strain was inoculated from agar slant into liquid sorbitol media. Sorbitol hard agar
(HA) plates were streaked with the same liquid culture. We made glycerol stocks of the
colonies obtained from HA plates.

Learn

Sorbitol HA plates exhibited growth but liquid culture did not show any turbidity.
Fungal contamination, seen in the liquid culture, was the probable reason.

### Cycle 4

Design

Unless we get growth in liquid culture, we would not be able to derive BC. We also knew
that HS media, not sorbitol media, is arguably the best at inducing BC overproduction in
*K. xylinus*. Thus, after consulting experts we planned to culture the bacteria
in
modified HS media of pH 4.5.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. BC production) of our work.

Test

The strain was inoculated from sorbitol HA plate into liquid modified HS media. HA
plates could not be prepared with this modified HS media owing to shortage of time.

Learn

The test phase is still going on.

## MCC Sheet

### Cycle 1

Design

As a back-up plan, we also tried to make a sheet of microcrystalline cellulose (MCC)
from the commercially available powdered form. Though it is not directly synthesized by
bacteria, such a sheet would help us with accomplishing our Proof of Concept goals. MCC
and BC are almost similar in their characteristics.

As a starting point, we tried to find out a suitable solvent for dissolving MCC powder.
Based on literature review, we shortlisted dimethyl sulfoxide (DMSO), 7% LiCl in
dimethyl acetamide (DMAc), and N-methylpyrrolidinone (NMP) as candidates for further
investigation. Drop casting was chosen as the characterization method of choice at the
initial phase because it saves wastage of resources.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. sheet production) of our work.

Test

MCC was mixed with these solvents and stirred overnight. To test whether it is possible
to form a mechanically strong film of MCC from the solutions, we performed drop casting
(on glass slide) at a small scale with each of the samples and also with different
sample volumes.

Learn

In general, quenching gave better results compared to annealing. Drops derived from
MCC+DMSO solution yielded a cracked surface upon quenching. Drops derived from MCC+NMP
solution gave an excessively flaky residue. Sheet derived from MCC+LiCl/DMAc solution
showed negligible presence of cracks, but it adsorbed moisture after it had returned to
room temperature. The BC sheet we desire to make cannot be flaky. Thus, MCC+NMP solution
was not subjected to further investigation.

### Cycle 2

Design

We sought to see whether the addition of a binder could influence the mechanical
integrity of the film and strengthen it.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. sheet production) of our work.

Test

We added 10% w/v polyvinyl alcohol (PVA) to MCC+DMSO as well as to MCC+LiCl/DMAc. Then,
we performed drop casting (on glass slide) using these solutions, all well above
100°C as
done before. One of the drop-casted films obtained from MCC+DMSO was subjected to atomic
force microscopy (AFM).

Learn

All the drop-casted films turned out to be patchy and unusually brownish. Surprisingly,
PVA is known to be strongly preferring the formation of mechanically strong films!
However, we found out that the heating regime (above 100°C) used for the samples had
exceeded the degradation temperature of PVA. It was the degradation of PVA that had
caused the charred appearance of the films.

Usage of vacuum oven was causing the film to come out of the substratum (i.e. glass
slide). It was interfering with the proper functioning of AFM.

MCC+LiCl/DMAc+PVA solution yielded highly flaky film, whereas MCC+DMSO+PVA film fairly
maintained its integrity. Thus, MCC+LiCl/DMAc+PVA solution was not subjected to further
investigation.

### Cycle 3

Design

The drop casting, thus, must be performed at a lower temperature.

Also, we ceased to use vacuum oven for drop casting purpose.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. sheet production) of our work.

Test

We performed drop casting (on glass slide) using MCC+DMSO+PVA below 100°C.

Learn

The drop-casted film turned out to be homogeneous, just as we desire. We have thus
finalized the optimal solvent, a beneficial additive, and the optimal conditions needed
to come up with a homogeneous, crack-less, non-charred MCC sheet.

## Hardware

### Cycle 1

Design

As the first step towards building a functional hardware as the implementation of our
novel strategy, we decided to coat the inner surface of a microchannel with MCC. It
would then serve as the proof of concept for a microchannel whose bore has been coated
with the functionalized bacterial cellulose (BC) filter we seek to develop. Important
aspects of this are the thickness of MCC coating, extent of uniformity in coat thickness
along the length of microchannel, durability of the coating etc.

We first planned to fabricate a microchannel of square cross section whose one side is
open. The Then, we can pour MCC+DMSO+PVA solution (suits our purpose, as determined by
our work on MCC sheet development) on it through the open face. Upon drying, we would
get a MCC coating on one of the faces. Lastly, a glass slab is to be used to seal the
open face. As glass is transparent, it would help us in probing into the flow
characteristics inside the microchannel by using fluorescent beads in future.

However, we anticipated many challenges with this design. First, only one of the faces
ultimately happens to have the MCC coating. This means that in the prototype we seek to
develop with functionalized BC sheet in place of MCC layer, only one side of the
microchannel would host the embedded proteins. Thus, filtration efficiency of the system
would decrease as most of the organophosphate molecules simply pass through the
microchannel without coming into contact with a protein molecule for even once. Second,
fabricating a microchannel in the clean room at requires special expertise and
prohibitive amount of resources, given the scarcity of time.

Thus, we changed our plan. We wanted some simpler system as our Proof of Concept. We
chose the tubes made up of polydimethylsiloxane (PDMS) whose diameter is in the order of
millimetres. These are readily available, cheap, inert, non-toxic, and non-flammable.
Being transparent, rheological studies would be quite easy to perform. Since PDMS is
almost always cured with some cross-linking agent, it shows time-dependent elastic
deformation. Low elastic modulus of PDMS enables these pipes to be flexible (this
property might be valuable while designing a working prototype of our project). We tried
to coat such pipes on the inner side with MCC.

Build

Neither construction of a DNA sequence nor its implementation into a chassis was
involved in this part (i.e. hardware development) of our work.

Test

We collected MCC+DMSO+PVA solution (as described previously) in a syringe and injected
that into a 10 cm long PDMS tube of about 5 mm inner diameter. The pipe was left to dry
in an orientation horizontal relative to ground.

Learn

Even after 48 hours, the solution inside pipe did not dry off. Neither did it settle on
the PDMS surface. It happened most probably because the PDMS surface is very
hydrophobic, whether MCC+DMSO+PVA solution is less so.

Thus, we planned to modify the PDMS surface in order to increase its hydrophilicity.

## References

1. Khairil Anuar INA, Banerjee A, Keeble AH *et al*. Spy&Go Purification of SpyTag-proteins
   using
   Pseudo-SpyCatcher to Access an Oligomerization Toolbox. *et al*>. 2019;10(1734). doi:
   10.1038/s41467-019-09678-w
2. Hatlem D, Trunk T, Linke D, Leo JC. Catching a SPY: Using the SpyCatcher-SpyTag and Related
   Systems for Labeling and Localizing Bacterial Proteins. *Int. J. Mol. Sci.*. 2019 Apr
   30;20(9):2129. doi: 10.3390/ijms20092129
3. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. Peptide Tag
   Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin. *Proc.
   Natl.
   Acad. Sci. USA*. 2012 Mar 20;109(12):E690-7. doi: 10.1073/pnas.1115485109
4. Datsenko KA, Wanner BL. One-step Inactivation of Chromosomal Genes in Escherichia coli K-12 using
   PCR Products. Proc. Natl. Acad. Sci. U. S. A. 2000 Jun 6;97(12):6640-5. doi: 10.1073/pnas.120163297
5. 5. Zhang C, Liu R, Xiang JF, Kang H. Dissolution Mechanism of Cellulose in
   N,N-Dimethylacetamide/Lithium Chloride: Revisiting through Molecular Interactions. The Journal of
   Physical Chemistry B. 2014 Jul;118:9507-9514. doi: 10.1021/jp506013c
6. 6. Bracic M, Mohan T, Kargl R, Griesser T, Hribernik S, KÃ¶stler S, Stana-Kleinscheka K,
   Fras-Zemljica L. Preparation of PDMS Ultrathin Films and Patterned Surface Modification with
   Cellulose. RSC Adv. 2014;4:11955-11961. doi: 10.1039/C3RA47380E

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# Team:IISc-Bangalore/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:IISc-Bangalore/Results

Results | IISc Bangalore

* [![](https://static.igem.org/mediawiki/2021/c/cd/T--IISc-Bangalore--images--team_iisc_logo_min.png)](https://2021.igem.org/Team%3AIISc-Bangalore)
* Project
* [Parts](https://2021.igem.org/Team%3AIISc-Bangalore/Parts)
* Wetlab
* Drylab
* Human Practices
* Team
* [Judging](https://2021.igem.org/Team%3AIISc-Bangalore/Judging)

* [Description](https://2021.igem.org/Team%3AIISc-Bangalore/Description)
* [Design](https://2021.igem.org/Team%3AIISc-Bangalore/Design)
* [Engineering](https://2021.igem.org/Team%3AIISc-Bangalore/Engineering)
* [Implementation](https://2021.igem.org/Team%3AIISc-Bangalore/Implementation)
* [Proof of Concept](https://2021.igem.org/Team%3AIISc-Bangalore/Proof_Of_Concept)
* [UN SDG](https://2021.igem.org/Team%3AIISc-Bangalore/Sustainable)
* [Safety](https://2021.igem.org/Team%3AIISc-Bangalore/Safety)
* [Contribution](https://2021.igem.org/Team%3AIISc-Bangalore/Contribution)

* [Results](https://2021.igem.org/Team%3AIISc-Bangalore/Results)
* [Lab Notebook](https://2021.igem.org/Team%3AIISc-Bangalore/Notebook)

* [Model](https://2021.igem.org/Team%3AIISc-Bangalore/Model)
* [Protein Structure
  Predictions](https://2021.igem.org/Team%3AIISc-Bangalore/Predictions)

* [Education and
  Communication](https://2021.igem.org/Team%3AIISc-Bangalore/Communication)
* [Integrated Human
  Practices](https://2021.igem.org/Team%3AIISc-Bangalore/Human_Practices)

* [Members](https://2021.igem.org/Team%3AIISc-Bangalore/Members)
* [Attributions](https://2021.igem.org/Team%3AIISc-Bangalore/Attributions)
* [Collaborations](https://2021.igem.org/Team%3AIISc-Bangalore/Collaborations)

Navigation dropdown

1. [Home](https://2021.igem.org/Team%3AIISc-Bangalore)
2. Project:
   1. [Description](https://2021.igem.org/Team%3AIISc-Bangalore/Description)
   2. [Design](https://2021.igem.org/Team%3AIISc-Bangalore/Design)
   3. [Engineering](https://2021.igem.org/Team%3AIISc-Bangalore/Engineering)
   4. [Implementation](https://2021.igem.org/Team%3AIISc-Bangalore/Implementation)
   5. [Proof of Concept](https://2021.igem.org/Team%3AIISc-Bangalore/Proof_Of_Concept)
   6. [UN SDG](https://2021.igem.org/Team%3AIISc-Bangalore/Sustainable)
   7. [Safety](https://2021.igem.org/Team%3AIISc-Bangalore/Safety)
   8. [Contribution](https://2021.igem.org/Team%3AIISc-Bangalore/Contribution)
3. [Parts](https://2021.igem.org/Team%3AIISc-Bangalore/Parts)
4. Lab work:
   1. [Results](https://2021.igem.org/Team%3AIISc-Bangalore/Results)
   2. [Lab notebook](https://2021.igem.org/Team%3AIISc-Bangalore/Notebook)
5. Drylab
   1. [Model](https://2021.igem.org/Team%3AIISc-Bangalore/Model)
   2. [Protein Structure Predictions](https://2021.igem.org/Team%3AIISc-Bangalore/Predictions)
6. Human Practices:
   1. [Education
      and Communication](https://2021.igem.org/Team%3AIISc-Bangalore/Communication)
   2. [Integrated Human
      Practices](https://2021.igem.org/Team%3AIISc-Bangalore/Human_Practices)
7. Team:
   1. [Members](https://2021.igem.org/Team%3AIISc-Bangalore/Members)
   2. [Attributions](https://2021.igem.org/Team%3AIISc-Bangalore/Attributions)
   3. [Collaborations](https://2021.igem.org/Team%3AIISc-Bangalore/Collaborations)
8. [Judging](https://2021.igem.org/Team%3AIISc-Bangalore/Judging)

Back to Top

# Results

Here, we have shown the results of the different parts - protein production and Bacterial
Cellulose or Microcrystalline Cellulose Sheet Production - of our project. Go through our
journey of experiments and their results as we went through multiple engineering design
cycles. We present several documented results we obtained from our experiments that show the
readers how far we had been able to progress.

Contents:

1. [Cloning and protein production](#lab1)
   1. [Cloning of C1 and C2](#cloning)
   2. [Expression tests](#expression)
   3. [Solubility Tests](#solubility)
   4. [C2(2.0)](#c2)
   5. [C2(3.0)](#c3)
2. [Making microcrystalline cellulose sheet from
   powder](#lab2)
3. [Overproduction of bacterial cellulose](#lab3)
   1. [Knocking out *celG* from *A.
      tumefaciens* for BC overproduction](#knock)
   2. [Using *K. xylinus* for BC
      production](#k)
4. [References](#ref)

## Cloning and protein production

For the functionalization aspect of our project, we have two constructs - C1 (SpyCatcher002-dCBD)
and C2 (OpdA-SpyTag002). This section mentions our efforts in order to produce, purify and
characterize these fusion proteins. We first cloned these modules in a suitable vector, carried out
some expression tests where we standardized the induction conditions, and then performed some
solubility tests as well, in order to check if these novel fusion proteins could be purified. We
were successful in figuring out suitable conditions for expression and purification of one of our
constructs, while it currently is a work-in-progress for the other construct.

### Cloning of C1 and C2

Constructs C1 (SpyCatcher002-dCBD) and C2 (OpdA-SpyTag002) were synthesized by GenScript and shipped
to
us after being cloned into pUC57-Kan vectors. We amplified the constructs using suitable primers in
order to carry out Gibson assembly, by performing PCR with KOD polymerase. Gel electrophoresis
results
are given below:

![](https://static.igem.org/mediawiki/2021/2/27/T--IISc-Bangalore--images--results--06.21_EtBr_C1-PCR.png)

Fig 1: C1 PCR (M = marker)

![](https://static.igem.org/mediawiki/2021/f/f5/T--IISc-Bangalore--images--results--06.22_EtBr_C2-PCR.png)

Fig 2: C2 PCR (M = marker)

In order to express our constructs in E. coli, we chose a pETMCN-T7 (KanR) vector,
because it has a
high
copy number and transcription using the T7 RNA polymerase can be strongly induced upon addition of
IPTG.

pETMCN-T7 was linearized by digestion with NdeI. PCR primers for amplification of C1 and C2 were
designed to have overlaps with pETMCN-T7 on either side of the NdeI site. Both the PCR amplified
constructs and the linearized vector were gel extracted. Gibson Assembly was performed on these
samples
to yield C1\_pETMCN-T7 and C2\_pETMCN-T7.

The above samples were transformed into *E. coli* DH5 \(\alpha\), inoculated into liquid
culture,
and
miniprepped.
Double digestion was performed using NheI and BglII to verify positive clones by insert release. Gel
electrophoresis results are given below:

![](https://static.igem.org/mediawiki/2021/3/3f/T--IISc-Bangalore--images--results--06.28_EtBr_C1-Gibson-Verify.png)

Fig 3: Verification of C1 clones using double digestion (sample 3 culture was
unsuccessful;
M = marker, EVC = double digested pETMCN-T7)

All C1 samples contained an insert of appropriate size (~1 kb) and were thus positive clones.
Samples
7
and 8 were chosen for further experiments (based on concentrations).

![](https://static.igem.org/mediawiki/2021/6/69/T--IISc-Bangalore--images--results--06.28_EtBr_C2-Gibson-Verify.png)

Fig 4: Verification of C2 clones using double digestion (M = marker, EVC = double
digested
pETMCN-T7)

C2 samples 3 and 5 showed anomalous bands, while the rest were positive clones (insert band around
~2kb). Samples 6 and 7 were chosen for further experiments.

### Expression Tests

The chosen positive clones were transformed into *E. coli* BL21(DE3) for protein production.
Secondary
liquid cultures were induced with 0.25 mM or 0.5 mM IPTG and incubated at 23°C for 6 hrs
or 30°C for
4
hrs. Cell pellets were lysed by boiling in SDS Laemmli buffer. SDS-PAGE was performed on all the
samples.

![](https://static.igem.org/mediawiki/2021/9/96/T--IISc-Bangalore--images--results--07.02_Coomassie_C1-Expression.png)

Fig 5: Expression tests for C1 (M = marker, U = uninduced)

No prominent band at ~25kDa (C1 molecular weight) could be observed for induced samples as compared
to
uninduced samples for any of the tested induction conditions.

![](https://static.igem.org/mediawiki/2021/3/34/T--IISc-Bangalore--images--results--07.02_Coomassie_C2-Expression.png)

Fig 6: Expression tests for C2 (M = marker, U = uninduced)

Brighter bands at ~75kDa (C2 molecular weight) could be observed for induced samples compared to
uninduced for 0.25 mM IPTG at both temperatures.

A Western blot was performed using anti-His primary antibody to detect C1, and C2 samples were taken
as
well.

![](https://static.igem.org/mediawiki/2021/d/db/T--IISc-Bangalore--images--results--07.09_Western_C1%2CC2-anti-His.png)

Fig 7: Western blot for C1 and C2 expression tests (M = marker, U = uninduced)

For C1, prominent bands were observed at ~25kDa, with induction being strongest for 0.5 mM IPTG
incubated at 30°C. This was taken to be the induction condition for a large-scale
culture. The same
conditions were finalized for C2 also since there was a prominent band at ~75kDa.

### Solubility Tests

Large scale *E. coli* BL21(DE3) cultures (50 mL each) were set up for both C1 and C2, with
induction
by
0.5 mM IPTG at 30°C. After cells were harvested, they were lysed and subjected to Ni-NTA
pulldown to
check solubility. SDS-PAGE was performed on the boiled samples taken from different fractions
(lysate,
pellet, supernatant, washed and bead-bound).

![](https://static.igem.org/mediawiki/2021/b/ba/T--IISc-Bangalore--images--results--07.10_Coomassie_C1%2CC2-Solubility.png)

Fig 8: Solubility test for C1 & C2 using Ni-NTA pulldown [M = marker, L = cell lysate, P
=
pellet, S = supernatant, BB = bead-bound (boiled), BE = bead-bound (eluted)]

Amounts of protein loaded were not normalized, and hence large blobs were observed in the pellet
fractions. The bead-bound fraction for C1 showed a large band at ~25kDa, which implied that C1 was
appreciably soluble. No such observation could be made for C2.

By this point, we had standardized the expression conditions for C1 and C2, and also verified that
C1
was indeed soluble. To verify the same for C2, two Western blots were performed on the C2 pulldown
samples, using anti-His and anti-GFP primary antibody respectively.

![](https://static.igem.org/mediawiki/2021/3/34/T--IISc-Bangalore--images--results--07.16_Western_C2-anti-GFP.png)

Fig 9: anti-GFP blot for C2 [M = marker, L = cell lysate, P = pellet, S =
supernatant,
BB =
bead-bound (boiled), NC = negative control (C1 BB fraction, 5 \( \mu \)L)]

![](https://static.igem.org/mediawiki/2021/c/c5/T--IISc-Bangalore--images--results--07.16_Western_C2-anti-His.png)

Fig 10: anti-His blot for C2 [M = marker, L = cell lysate, P = pellet, S =
supernatant,
BB = bead-bound (boiled), PC = positive control (C1 BB fraction, 5 \( \mu \)L)]

In the anti-GFP blot, a prominent band at ~75kDa could be observed, but there were a lot of other
bands
too, and similar lower molecular weight bands were observed for the anti-His blot too. This
suggested
that there was a lot of non-specific antibody binding, contamination, or protein degradation in case
of
C2. This was repeated for more dilute antibody treatments, but results looked similar, which led us
to
believe that C2 was indeed getting degraded.

The pulldown of C2 was performed again with an anti-GFP nanobody coupled to GST tagged
glutathione-sepharose beads. SDS-PAGE was performed on the samples.

![](https://static.igem.org/mediawiki/2021/d/d3/T--IISc-Bangalore--images--results--07.30_Coomasie_C2-GFP-Nb.png)

Fig 11: Solubility test for C2 using anti-GFP pulldown [M = marker, U = uninduced, L =
cell
lysate, P = pellet, S = supernatant, BB = bead-bound (boiled)]

Prominent bands at ~37kDa were observed corresponding to the nanobody, but none at ~75kDa, which
implied
that pulldown was unsuccessful. We suspected that C2 might not be properly soluble and hence might
not
have been interacting with the anti-GFP nanobody tagged beads. We thus decided to modify the C2
construct.

### C2(2.0)

In order to make C2 less bulky, we decided to take out the sfGFP module. This was taken into account
in
the design phase of the project, and KpnI restriction sites had been included on either end of the
sfGFP
CDS. C2\_pETMCN-T7 was digested with KpnI followed by ligation to yield C2(2.0)\_pETMCN-T7.

![](https://static.igem.org/mediawiki/2021/1/13/T--IISc-Bangalore--images--results--Map_C2%282.0%29.png)

Fig 12: Schematic map of C2(2.0)

Positive clones were identified using insert release by digestion with BamHI and BglII.
Transformation
was done into *E. coli* BL21(DE3) followed by Ni-NTA pulldown as before.

![](https://static.igem.org/mediawiki/2021/1/12/T--IISc-Bangalore--images--results--08.03_Coomassie_C2%282.0%29-Ni-NTA.png)

Fig 13: Solubility test for C2(2.0) using Ni-NTA pulldown [M = marker, U = uninduced, L
=
cell lysate, P = pellet, S = supernatant, BB = bead-bound (boiled)]

No prominent band at ~50kDa (C2(2.0) molecular weight) could be observed.

Pulldown was performed again using SpyDock linked Ni-NTA beads.

![](https://static.igem.org/mediawiki/2021/f/f1/T--IISc-Bangalore--images--results--08.11_Coomassie_C2%282.0%29-SpyDock.png)

Fig 14: Solubility test for C2(2.0) using SpyDock pulldown [M = marker, U = uninduced, L
=
cell lysate, P = pellet, S = supernatant, BB = bead-bound (boiled)]

Although a prominent band could be observed above ~15kDa (SpyDock molecular weight), none could be
observed at ~50kDa. Till this point, we had not been able to verify solubility of either C2 or
C2(2.0).
We suspected that the hydrophobic signal peptide attached to OpdA might be leading to aggregation,
so we
decided to modify C2 yet again.

In the meanwhile, we also decided to conduct some assays on the crude extract of the large-scale
secondary cultures. The assays we did were the Bradford Assay and the assay of the OpdA domain using
Coumaphos. We also decided to grow the bacteria in both LB and Terrific Broth (TB), because a short
literature review showed that some people have used TB as the growth medium for the secondary
culture for the OpdA production

We used both the C2 and the C2 (2.0) constructs for this purpose.

For this, we harvested the protein from the cells. On the crude cell extract, we conducted the
Bradford assay, using BSA as the reference for the standard curve. The standard curve is shown
below:

![](https://static.igem.org/mediawiki/2021/1/1c/T--IISc-Bangalore--images--results--some_curve.png)

| Sample | Adjusted Absorbance | Estimated Concentration (mg/mL) |
| --- | --- | --- |
| LB EV | 0.9034 | 0.5937 |
| LB C2 | 0.8659 | 0.5445 |
| LB C2 (2.0) | 0.8607 | 0.5377 |
| LB NT | 0.9296 | 0.6281 |
| TB EV | 0.9449 | 0.6482 |
| TB C2 | 1.0485 | 0.7841 |
| TB C2 (2.0) | 0.729 | 0.3648 |
| TB NT | 1.109 | 0.8636 |

In the above table, EV refers to Empty Vector samples. NT refers to non-transformed samples.

Surprisingly, the non-transformed TB culture had the highest protein concentration, while the C2
(2.0) cultures had the lowest in their corresponding set, be it LB or TB.

For Enzyme assay, we dissolved Coumaphos in Ches Buffer and used it as a substrate. The product,
chlorferon has an absorption peak at 348 nm. The absorbance values of the different samples are as
follows:

| Culture Medium | EV | C2 | C2 (2.0) | NT |
| --- | --- | --- | --- | --- |
| LB 1 | -0.189 | 0.111 | 0.188 | Used as Blank |
| LB 2 | 0.824 | 0.314 | 0.09 | Used as Blank |
| TB 1 | 0.241 | 0.647 | 1.599 | Used as Blank |
| TB 2 | 1.333 | 1.240 | 0.683 | Used as Blank |

LB 1 and LB2 represent replicates, so do TB1 and TB2 for the corresponding cultures.

A very broad trend that was easily seen was that enzyme production seemed to be higher in cultures
grown in TB than those grown in LB. So, we can continue using TB as the growth medium for our
protein's expression.

We normalized the absorbance of the test samples at 348 nm (corresponding to lambda max of
chlorferon, the
degradation product of assay substrate coumaphos) with their respective protein concentrations as
deduced from the Bradford assay. In OpdA assay, NT was used as the blank (thus, futile to deal with
its absorbance, be it normalized or not). The normalized absorbance (in arbitrary units) values are
tabulated below:

| Culture Medium | EV | C2 | C2(2.0) | NT |
| --- | --- | --- | --- | --- |
| LB 1 | -0.31834 | 0.20386 | 0.34964 | Used as Blank |
| LB 2 | 1.38791 | 0.57668 | 0.16738 | Used as Blank |
| Average of the values | 0.53479 | 0.39027 | 0.25851 | - |
| TB 1 | 0.37180 | 0.82515 | 4.38322 | Used as Blank |
| TB 2 | 2.05646 | 1.58143 | 1.87226 | Used as Blank |
| Average of the values | 1.21413 | 1.203 | 3.1277 | - |

Here '1' and '2' simply denote two replicates of the OpdA assay with the samples mentioned, in
chronological order.

To understand the trend exhibited by the average values, a bar chart was plotted:

![](https://static.igem.org/mediawiki/2021/f/f6/T--IISc-Bangalore--images--POC--Proof_of_Concept.png)

The normalized absorbances of the degradation product can be considered as a measure for enzyme
activity in the crude cell extract. As can be observed from the graph, when grown in LB, the enzyme
activity seemed to decrease from the Empty Vector control to C2 to C2 (2.0). However, when grown in
Terrific Broth (TB), while there is almost no change in enzyme activity between C2 and Empty Vector
control, there is a huge increase in enzymatic activity in case of C2 (2.0).

For our interpretation of these results, please visit the [Proof of Concept](https://2021.igem.org/Team%3AIISc-Bangalore/Proof_Of_Concept) page.

### C2(3.0)

In order to take out the OpdA signal peptide, a PCR was performed on C2(2.0)\_pETMCN-T7 to amplify
C2(2.0) minus the N-terminal signal. This PCR fragment was cloned into an empty pETMCN-T7 vector to
yield C2(3.0)\_pETMCN-T7.

![](https://static.igem.org/mediawiki/2021/6/6f/T--IISc-Bangalore--images--results--Map_C2%283.0%29.png)

Fig 15: Schematic map of C2(3.0)

We are in the "Build" phase of this engineering cycle. We are yet to clone the bacteria into
BL21(DE3) cells. So, the progress on this is not significant enough to give us any conclusive
findings

## Making microcrystalline cellulose sheet from MCC powder

We also tried to make a sheet of microcrystalline cellulose (MCC) from the commercially
available
powdered form. Powder of MCC was purchased from Sigma Aldrich & Co. Initially, we started out on
finding a suitable solvent for dissolving MCC powder. Although data is available in literature
in
this regard, they are not exhaustive and are often ambiguous. Based on literature review, we
decided
to focus on DMSO (dimethyl sulfoxide), NMP (N-methyl pyrrolidinone and a solution of 7% LiCl in
DMAc
(dimethyl acetamide) as probable solvents for further investigation.

MCC was mixed with these solvents and stirred on a magnetic stirrer overnight. The results were
as
follows:

| Solution | Solute | Solvent | Stirring Speed | Results | Inference |
| --- | --- | --- | --- | --- | --- |
| Cellulose + DMSO | 1 g MCC powder | 10 mL DMSO | 450 rpm | Maximum cloudiness | Maximum solubility of MCC |
| Cellulose + 7% LiCl in DMAc | 1 g MCC powder | 7% LiCl in DMAc | 450 rpm | Intermediate cloudiness | Intermediate solubility of MCC |
| Cellulose + NMP | 1 g MCC powder | 10 mL NMP | 460 rpm | Minimum cloudiness | Minimum solubility of MCC |

![](https://static.igem.org/mediawiki/2021/e/e7/T--IISc-Bangalore--images--results--PCRLab_Fig1-1.jpg)

![](https://static.igem.org/mediawiki/2021/c/c5/T--IISc-Bangalore--images--results--PCRLab_Fig1-2.jpg)

![](https://static.igem.org/mediawiki/2021/2/21/T--IISc-Bangalore--images--results--PCRLab_Fig1-3.jpg)

To test whether it was possible to form a mechanically strong film of bacterial cellulose from
the
solutions in these solvents, we decided to perform a standard test used by materials engineers
for
understanding this - drop casting. This small scale test saves wastage of resources by doing a
random hit-and-trial with huge volumes of samples. A drop of the sample is spread out over a
glass
slide and heated at a temperature about 20 oC below its boiling point, and then subjected to a
variety of treatment conditions. The film formed on the glass slide is observed.

![](https://static.igem.org/mediawiki/2021/2/22/T--IISc-Bangalore--images--results--PCRLab_Fig2.jpg)

Drop-castings were performed with different sample volumes. Since MCC was most soluble in DMSO
among
the three solvents, we decided to investigate the influence of different heating durations on
the
integrity of the film formed from this solution. The results of our drop-castings using the
above
mentioned solvents are enlisted below:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Label | Solvent | Temp. used | Volume used | Time of heating | Observations |
| DMSO 1 | DMSO | 170°C | 1 mL | 30 s | Cracked when quenched |
| DMSO 2 | DMSO | 170°C | 0.5 mL | 20 s | Large cracks; Highly non-uniform thickness |
| DMSO 3 | DMSO | 170°C | 1 mL | 35 s | Large cracks |
| NMP 1 | NMP | 175°C | 0.6 mL | 20 s | Microcracks on visual examination after annealing. Excessively flaky. |
| LiCl/DMAc 1 | 7% LiCl in DMAc | 140°C | 1 mL | 45 s | Negligible presence of cracks. A few small bubbles; absorbed moisture after annealing; after heating for more than 850 s, turned yellow |
| LiCl/DMAc 2 | 7% LiCl in DMAc | 140°C | 0.5 mL | 420 s | Almost no cracks; bubbles; absorbed moisture |

![](https://static.igem.org/mediawiki/2021/3/3f/T--IISc-Bangalore--images--results--PCRLab_Fig3.jpg)

Next, we sought to see if the addition of a binder like PVA (polyvinyl alcohol) could influence
the
mechanical integrity of the film and strengthen it. We tried to carry out drop-castings using
the
same solutions, but with 10 % w/v PVA added to it. Since quenching gave better results
previously,
all the samples were quenched on a pool of acetone after heating to evaporate the solvent.
Temperatures for heating were same as before. However, we found that all the drop-casted films
were
patchy and flaky and had turned brownish, except for the solution containing DMSO as solvent,
which
formed a homogenous sheet, albeit with a brownish hue.

![](https://static.igem.org/mediawiki/2021/6/6d/T--IISc-Bangalore--images--results--PCRLab_Fig4.jpg)

Fig: Q denotes Quenching, A denotes annealing. N indicates normal drying (by leaving
it
out in
air)
while O denotes drying in hot air oven.

Having been slightly surprised at these results, we were intrigued to find out the possible
cause(s)
behind this. PVA is known to be strongly preferring the formation of mechanically strong films.
The
results therefore seem counter-intuitive at the first glance. However, a survey of existing
literature and the materials data sheets provided by companies led us to conclude that the
heating
regime used for most of the samples had exceeded the degradation temperature of PVA, while the
heating temperature for DMSO was slightly lower than the average degradation temperature. The
brownish colour was most likely imparted due to the influence of the degradation products of
PVA.

We therefore decided to redo our experiment for PVA by using a lower temperature. Although this
would prolong the heating duration, we expected to see a stable film being formed, devoid of the
brownish hue of the previous case. Our hypothesis was indeed validated in the next experiment

|  |  |  |
| --- | --- | --- |
| Solution | Heating | Observations |
| DMSO + 10% PVA + MCC | Heated on Hot Plate gradually till 90°C for 8.5 min and then rapidly quenched | Homogenous sheet |

We have thus tried to optimise the conditions for making MCC sheet from commercially available
powder. Further studies need to be done by drop-casting on a block of PDMS or inside a PDMS tube
instead of a glass slide. Detailed studies need to also be performed on the interesting solvent
system of 7% LiCl in DMAc, because the film formed in this case absorbs moisture due to the
presence
of hygroscopic LiCl and spontaneously gets healed of any cracks present.

## Overproduction of bacterial cellulose

### Knocking out *celG* from *A. tumefaciens* for BC overproduction

We decided to knock out the celG gene from the chvB- mutant A. tumefaciens C58 via FLP-FRT
recombination.

For this purpose, the lambda red recombination cassette of pKD46(AmpR) was amplified using
gradient PCR and put under tetR promoter in pST-KT. We also planned to amplify the
complement1-FRT-CamR-FRT-complement2 fragment which would replace the part of the *celG*
sequence
we wished to remove. The complement1 and complement2 are DNA sequences that are complementary to
the regions upstream and downstream of the portion we want to knock out.

We were able to successfully amplify the lambda red recombination cassette. Based on the gel
images, we can see that the PCR product amount was highest at 50°C. No product was obtained
at
all at 52°C and 58°C.

We couldn't amplify the FRT-complements fragment. Based on the gel images, we believe this is
probably due to the formation of primer-dimers.

![](https://static.igem.org/mediawiki/2021/1/17/T--IISc-Bangalore--images--results--lambda_red.jpg)

Gel after amplifying lamdba Red Recombination Cassette (from left to right),
Ladder and Gradient PCR products for temperatures 50°C, 52°C, 54°C,
56°C, 58°C

![](https://static.igem.org/mediawiki/2021/c/c3/T--IISc-Bangalore--images--results--SP_lab.jpg)

Unsuccessful gel showing Primer-Dimer bands at bottom for FRT fragments which
could not be amplified (from left to right), Gradient PCR products for temperatures
50°C, 52°C, Ladder, Gradient PCR products for 54°C, 56°C, 58°C,
60°C

The bacterial cultures were received from Prof. Ann G. Matthysse. However, we could not revive
the bacteria on the LB Agar plates despite following the instructions sent by Prof. Matthysse.
She said that this might be because the cultures sent were too old.

We are still figuring out how to proceed with this part of the plan. In the meanwhile, we
decided to go for an alternate strategy for producing bacterial cellulose, the results of which
have been provided at the end.

### Using *K. xylinus* for BC production

To produce Bacterial Cellulose sheet, we decided to grow a culture of *Komagataeibacter
xylinus* (also
known as *A. xylinum*) as it is known to overproduce bacterial cellulose when grown in
the
Hestrin-Schramm (HS) culture medium at 28°C, as per the paper Keshk, S. M. A. S. (2006)

Thus, we decided to try growing the bacteria in the HS medium.

We first ordered an Agar slant of the ATCC 11142 strain of the bacteria from the National
Chemical
Laboratories in Pune.

As per the HS media preparation protocol of the 2020 UCSC iGEM Team, we prepared the HS medium
and
inoculated our bacteria into that. However, even after 3-4 days, no growth was observed, which
was
concerning given that the bacterium has a generation time of about 3 days.

We then decided to try the medium, which was recommended by NCL, Pune to be used to revive or
store
the culture. This medium, unlike the HS medium, has no buffer components and has sorbitol
instead of
glucose as the carbon source. This medium, we hereafter refer to as the "Sorbitol medium".

We tried to grow our bacteria in the Sorbitol medium, and the bacteria successfully grew in the
medium.

![](https://static.igem.org/mediawiki/2021/2/22/T--IISc-Bangalore--images--results--lab3_1.jpg)

Figure 1: 2 bacterial culture plates in Sorbitol Hard Agar medium with the same
bacterial culture in LB Agar as a reference. As can be seen, there is bacterial growth
in
the
Sorbitol Hard Agar plates and zero growth on the LB Agar plates.

However, there were a host of other complications:

1. Being an extremely general medium containing nothing but yeast extract, sorbitol medium is
   highly prone to contamination. Many of our samples and even some of the medium prepared by
   us
   got contaminated (Figure 2 and Figure 3).

![](https://static.igem.org/mediawiki/2021/7/78/T--IISc-Bangalore--images--results--lab3_2.jpg)

Figure 2: Fungal Contamination

![](https://static.igem.org/mediawiki/2021/8/86/T--IISc-Bangalore--images--results--lab3_3.jpg)

Figure 3: Contaminated Culture in Sorbitol Liquid Medium

2. Bacterial Cellulose production was not observed in Sorbitol medium as well despite the
   bacteria
   growing. This is probably because Sorbitol is not such a good carbon source for cellulose
   production.

After several unsuccessful attempts at growing the bacteria in HS media or getting to produce
bacterial cellulose in Sorbitol medium, we talked to Dr. Mudrika Khandelwal and Dr. Shivakalyani
Adepu from The Cellulose Group at the Department of Materials Science and Metallurgical
Engineering
of IIT Hyderabad.

Based on their suggestion and using the protocol provided in their paper (Adepu, S. 2020), we
changed the composition of the HS medium to have 3.4 g of Na2HPO4 instead of 2.7 g and pH 4.5
instead of 6. We then tried to grow our bacteria in this medium and we were successful!

In fact, we inoculated two cultures, one in the HS medium and one in the Sorbitol medium. While
the
one in the Sorbitol medium got contaminated as shown by the rapid increase in OD600,
the
one in HS
medium grew at a slow and steady state, showing that Sorbitol medium is much
more prone to contamination.

However, we now tried to follow their protocol to setup secondary cultures for bacterial
cellulose
production. But, even 2 weeks later, there were no changes observed in the secondary culture
(Figure
4).

![](https://static.igem.org/mediawiki/2021/8/86/T--IISc-Bangalore--images--results--lab3_4.jpg)

Figure 4: Secondary culture of *K. xylinus* in modified HS medium

## References:

1. Keshk, S. M. A. S. "Physical properties of bacterial cellulose sheets produced in presence
   of
   lignosulfonate." *Enzyme and Microbial Technology* 40.1 (2006): 9-12.
   [doi.org/10.1016/j.enzmictec.2006.07.038](https://doi.org/10.1016/j.enzmictec.2006.07.038)
2. Adepu, S., & Khandelwal, M. (2020). Ex-situ modification of bacterial cellulose for
   immediate
   and sustained drug release with insights into release mechanism. *Carbohydrate
   Polymers*,
   249,
   116816. [doi.org/10.1016/j.carbpol.2020.116816](https://doi.org/10.1016/j.carbpol.2020.116816)

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