iGEM 2023 — Team uchicago

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# Notebook

“We are what we repeatedly do”
 - Aristotle

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Details of our wet lab work can be found in the following document. For
information on the protocols used, check out our
[contribution](https://2023.igem.wiki/uchicago/contribution)
page.

---

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# Engineering

“During a intense period of lab work the outside world
vanishes and the obsession is total.”
- Leon M. Lederman

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The biological system of E. coli was engineered and re-wired to produce our
target enzyme. This engineering involved designing changes to its genetic
make-up through the introduction of a constructed plasmid that codes
information on the chosen transaminases. By using the engineering cycle we
were able to define our expected outcomes and build constraints. We used the
phases of the cycle to reach as close as possible to our ideal outcome while
also reflecting on ways to improve our experimental design. The end of each
engineering cycle prompted the start of another cycle or provided
suggestions for improvements to be implemented in the future.

{% endblock %} {% block section\_links %}- [Cycle 1: Cloning in pET-15b](#cycle1)
- [Cycle 2: Test Expressions](#cycle2)
- [Cycle 3: UV-Vis](#cycle3)
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## Cycle 1: Cloning in pET-15b

---

### Design

To express our transaminase proteins, we needed a host vector backbone
that was well characterized and capable of producing high copy numbers.
Based on availability of vectors in our host lab we chose to clone our
proteins into the pET-15b vector. The vector allowed for IPTG induced
expression which was an effective way to control expression of the
enzymes. The easy access to and the adaptability of the vector to serve
our purpose of protein expression made it suitable to host our
transaminase sequence.

### Build

In accordance with our initial plasmid design, we planned to clone three
transaminases (AATA, HHTA, and CVTA) into the pET-15b vector. This was
accomplished through restriction enzyme digestion and Gibson assembly.
We then transformed our plasmid into DH5-alpha and BL21 competent cells
and confirmed successful cloning by full plasmid sequencing.

### Test

After successfully cloning into pET-15b, we started test expressions,
varying IPTG induction concentrations, and incubation temperatures and
durations in order to determine the best conditions for expression. We
performed pull-down assays using Ni-NTA resin, followed by SDS-PAGE gel
electrophoresis to see if our transaminases were being expressed.
However, initial results were inconsistent across different gels, so we
performed a western blot to more conclusively determine whether or not
protein was being expressed. This indicated a complete absence of any
protein expression.

![western blot](https://static.igem.wiki/teams/4710/wiki/engineering-success/es-fig1.png)

Figure 1: Western blot of all three transaminases after expression
for 5 hours at 30˚C and a nickel pulldown assay. Pos Ctrl: dsRed-Max
red fluorescent protein. U: uninduced. I: induced with 1mM IPTG

### Learn

We believe that this was due to an indel in the lacI gene in our pET-15b
vector, which was revealed by the results of plasmid sequencing. The
lacI gene codes for the lac repressor protein which regulates the
expression of our transaminases. The indel in the lac repressor was
likely responsible for the lack of expression with and without IPTG.

![full plasmid seq](https://static.igem.wiki/teams/4710/wiki/engineering-success/es-fig2.png)

Figure 2: Screenshot of full plasmid sequencing results showing an
indel in the LacI gene

Realizing that modifying protein expression conditions wouldn't rectify
this vector-related problem, we decided to reclone our constructs into a
different vector. We chose pQE81XN, the vector that hosted our positive
control, for the recloning because, as seen in Figure 1, our positive
control was expressing well, and so we were confident that recloning
into this vector could lead to protein expression.

## Cycle 2: Test Expressions

---

### Design

Based on what we had learned from cycle one, we redesigned our plasmids,
planning to clone into the pQE81XN vector that we knew was capable of
protein expression due to how well our positive control expressed in
test expressions.

### Build

We cloned our transaminase inserts into pQE81XN and then transformed our
plasmids into DH5-alpha and BL21 competent cells, using our same
protocols from cycle 1 but integrating what we had learned from this
previous iteration of the cloning cycle.

### Test

After recloning into the pQE81XN vector, we again perform test
expressions in order to optimize protein expression. We induced cells
with 1mM IPTG at 16C for 4 hours and 16 hours. Initial test expressions
([Figure 3](#figure3)) revealed that AATA expressed well and
HHTA expressed sparingly, while CVTA did not express at all.

![protein gel](https://static.igem.wiki/teams/4710/wiki/engineering-success/es-fig3.png)

Figure 3: SDS-PAGE gel electrophoresis of transaminases after
expression in BL21 cells, Hi-NTA pulldown and denaturation at 90C.
Pos Ctrl: Ds-Red-M in trimer form. U: Uninduced. I: Induced with 1mM
IPTG.

### Learn

Our new pQE81XN plasmids expressed AATA and HHTA, but CVTA still did not
express. While we tried to understand why the CVTA expression failed, we
were able to optimize expression of AATA and HHTA. Test expressions
showed that lower temperatures and longer incubations produced better
expression than higher temperatures and shorter incubations. This
permitted us to move forward with larger-scale expressions of these two
transaminases.

## Cycle 3: Preparing UV-Vis Assay

---

### Design

To characterize the enzymatic activity of our transaminases, we decided
to take advantage of the unique absorption spectra of the intermediates
formed throughout the reaction. To ensure that the only absorption data
we were collecting was a result of the transamination reaction and not
the substrate's inherent absorption, we aimed to normalize the data with
the absorption of individual substrates at the same concentration.

### Build

To conduct the following, we used a UV-Vis spectrophotometer and
prepared 500 uL reactions containing a fixed amount of enzyme and
substrate.We visualized the formation of intermediates by taking
absorption readings after the addition of every substrate and by
tracking the same through time. To prevent unbound PLP from affecting
results, we desalted the enzymes to remove any unbound PLP.

### Test

The enzyme without any substrates is expected to form a peak at 411 nm
as a result of the covalent binding of a protonated PLP to the enzyme’s
active site as a coenzyme. However, when we first conducted the assay,
we observed no visible peaks at 411 nm but instead a peak at ~ 350 nm as
seen in [Figure 4A](#figure4). This corresponds to
deprotonated PLP and could lead to an inactive active site.

![UV-Vis Spectrum](https://static.igem.wiki/teams/4710/wiki/engineering-success/es-fig4.png)

Figure 4: A: UV-Vis spectrum of AATA incubated with 0.1mM PLP from
the 100mM PLP in HEPES stock. B: UV-Vis spectrum of 0.1mM PLP in TBS
Buffer. C: UV-Vis spectrum of 100mM PLP in HEPES

### Learn

To troubleshoot this problem, we decided to check the absorbance of our
stock solution itself. The stock solution used for this preliminary test
was from a 100mM PLP in HEPES stock which as seen in Fig 4C showed
unreliable absorbance spectrum causing us to doubt the integrity of the
stock solution. We also had a secondary stock of PLP in the TBS buffer
which we tested on UV-Vis and observed expected absorbance (Fig 4B). We
were able to infer that PLP degrades over time when stored in HEPES but
is relatively more stable in TBS kept at 4˚C in the dark. Using this
learning, we were able to conduct a new assay by incubating the enzymes
with PLP from the stock in the TBS buffer. This cycle was a success as
seen in Figure 5 since we were able to show the correct covalent binding
of the enzyme to PLP.

![UV-Vis Spectrum](https://static.igem.wiki/teams/4710/wiki/engineering-success/es-fig5.png)

Figure 5: UV-Vis Spectrum of three trials of HHTA (at 0.8 mg/mL) and
three trials of AATA (at 1 mg/mL) in HEPES buffer bound to PLP to
form an internal aldimine with a distinct peak at 411 nm.

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# Experiments

“Experiment is the sole judge of the validity of any idea.”

- Richard P. Feynman

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A variety of experiments were performed over the course of our project, and
these can be divided into 3 phases:
**cloning**, **protein expression & purification**, and
**UV-Vis characterization** of our transaminases.

#### Cloning:

* Restriction enzyme digestion
* Agarose gel electrophoresis
* Gibson assembly
* Transformation: DH5-alpha and BL21 competent cells
* Glycerol stock preparation

#### Protein Expression & Purification

* IPTG induction
* Cell lysis with BPERII
* Purification with Ni-NTA resin
* SDS PAGE gel electrophoresis

#### UV-Vis Characterization:

* Utilization of UV-Vis assays to assess the enzymatic
  activity of our transaminases
* Measurement of absorbance of transamination
  intermediaries

See more of our
[protocols](https://2023.igem.wiki/uchicago/contribution)
See more of our
[lab notebook](https://2023.igem.wiki/uchicago/notebook)
See more of our
[experimental results](https://2023.igem.wiki/uchicago/results)

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# Results

“What is research but a blind date with knowledge?”

- Will Harvey

{% endblock %} {% block section\_links %}- [Phase 1: Cloning](#cloning)
- [Phase 2: Protein Expression and Purification](#expression)
- [Phase 3: Characterization of Enzymatic Activity](#characterization)
- [Recommendations For Future Studies](#recommendations)
- [Supplementary Data](#supplementary)
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## Phase 1: Cloning

---

We successfully cloned our transaminases into the pQE81XN vector which
hosted our positive control as indicated by full plasmid sequencing.
However, while conducting test expressions to optimize conditions for
maximal protein yield, we noticed that irrespective of the condition,
CVTA failed to express. While we tried to understand why the CVTA
expression failed, we decided to move forward with the large-scale
expressions of HHTA and AATA.

## Phase 2: Protein Expression and Purification

---

Since test expressions of AATA and HHTA were successful, we scaled up to
1 liter expressions by inoculating 1 L TB with 50 ml overnights of our
transaminase sequences in BL21 cells and inducing expression with 1mM
IPTG. Using Ni-NTA resin binding, we purified our His-Tagged
transaminases and quantified the purified transaminases using a Bradford
assay.

The results indicated that a 1 L culture of transformed E. coli yields
approximately 29.77 mg of AATA and 2.1 mg of HHTA. We stored the enzymes
in the TBS buffer with pyridoxal 5’-phosphate (PLP, cofactor to keep it
stable as a homodimer) at - 80˚C. However, we noticed that the
freeze-thaw led to the enzyme precipitating. We reconducted the large
scale expression and purification with HEPES buffer (with 0.1 mM PLP)
instead of TBS and stored the enzymes in the dark at 4˚C. We were also
able to optimize the purification process to decrease protein lost at
each step and obtained roughly 30 mg of AATA and 6.5 mg of HHTA from a 1
L media culture ([Figure 4](#figure4)).

![](https://static.igem.wiki/teams/4710/wiki/results/results-fig1.png)

Figure 1: SDS-PAGE gel electrophoresis of AATA and HHTA after
expression in BL21 cells with 1mM IPTG, Hi-NTA pulldown and
denaturation at 90C. E1: Elution 1. E2: Elution 2

## Phase 3: Characterization of Enzymatic Activity

---

### Background

The Ultraviolet–visible spectroscopy (UV-VIS) assay relies on the
formation of 4 intermediates involved in the transamination of the
substrate, each with a unique absorbance. This makes the reaction
trackable using UV-vis spectroscopy, and by measuring the intensity of
the absorbance peak, we can determine the activity of the reaction.

First, PLP covalently binds to the transaminase through an imine linkage
with a lysine residue, forming a Schiff base. This structure is known as
the internal aldimine, and it has an absorbance of 411 nm. An aspartate
residue in the active site of the enzyme protonates the nitrogen in
PLP’s pyridine ring, creating an electron sink for further reaction.

When the amine donor is added, it displaces the lysine residue to form
the external aldimine, which has an absorbance of 330 nm. The external
aldimine reacts with water to form the ketone byproduct and PMP, which
has an absorbance at 314nm. The addition of the amine acceptor reforms
the external aldimine, which then reacts to form the final amine
product. The internal aldimine is regenerated, completing the catalytic
cycle.

![](https://static.igem.wiki/teams/4710/wiki/results/results-figure2.png)

Figure 2: A. Scheme of key intermediates of a transaminase reaction.
B. UV-vis spectra of intermediates, using S-MBA as the amine donor
and pyruvate as the amine acceptor, collected by M. Saajid
(Campopiano Group, U of Edinburgh). Colored lines correspond to the
four intermediates depicted in Figure 2A.

The covalent binding of PLP to the transaminase dimer is essential for
its enzyme activity and to ensure that there was enough PLP to
covalently bind to the purified transaminases, we incubated the enzymes
with excess PLP before desalting it to remove excess PLP. This ensured
that the UV-VIS spectrum wasn’t influenced by unbound PLP. Initial
trials indicated that the transaminases worked best at concentrations
close to 1 mg/mL. After concentrating HHTA and AATA, we performed the
assay at 1 mg/mL of AATA and 0.8 mg/mL of HHTA (based on A280
absorbance). Both amine acceptor and amine donors were added to a final
concentration of 2.5mM each. For AATA, L-phenylalanine was used as the
amine donor while for HHTA, L-kynurenine was used as the amine donor.
Due to a scarcity of previous studies on AATA, we tested two known amine
acceptors that work on a variety of transaminases, sodium pyruvate and
sodium phenylpyruvic acid as our control acceptors. HHTA is reported to
accept sodium phenylpyruvic acid as an amine acceptor. For the
production of 3,5-diiodo-L-tyrosine (“mini thyroxine”), our target
product, we used the alpha keto acid of the compound,
3,5-diiodo-phenylpyruvic acid as our test amine acceptor.

### Covalent binding of PLP to transaminases

As described above, when PLP covalently binds to the enzyme, it forms an
internal aldimine ring. To detect this correct binding, we can measure
the absorbance at 411 nm which corresponds to the formation of an
internal aldimine. As seen in Figure 3, there is a distinct peak at 411
nm for both AATA and HHTA indicating the
**correct covalent bonding of PLP, as a cofactor, to the
enzymes.**

![](https://static.igem.wiki/teams/4710/wiki/results/results-figure3.png)

Figure 3: UV-VIS Spectrum of three trials of HHTA (at 0.8 mg/mL) and
three trials of AATA (at 1 mg/mL) in HEPES buffer bound to PLP to
form an internal aldimine with a distinct peak at 411 nm.

### Enzymatic activity of HHTA

While we were successfully able to collect decipherable data on the
enzymatic activity of AATA, our reading on the enzymatic activity of
HHTA was skewed by the nature of the substrates required for the
reaction in HHTA. L-kynurenine, the amine donor for HHTA, is a colored
compound in solution and has significant absorption in the 300-600 nm
range. It is also likely that our source of L-kynurenine had degraded
because every reading of samples containing the substrate showed
inconsistencies (See Supplementary Data). Due to this, we were unable to
produce a reliable characterization of the enzymatic activity of HHTA.
Moving forward, we would like to source different amine donors or re-try
l-kynurenine with a more reliable methodology of activity assay, like
mass-spectrometry.

### Enzymatic activity of AATA with phenylpyruvic acid

Since we were unable to find reliable characterizations of
transamination reactions with AATA we chose to try commonly accepted
amine acceptors that are known substrates to multiple classes of
transaminases. One such amine acceptor is phenylpyruvate. When we
carried out this assay using L-phenylalanine as the amine donor, we
noticed that it seems, as seen in Figure 4, as though
**AATA was successfully able to undergo the enzymatic cycle**
and complete the transfer of the amine group. The evidence that points
to this is seen by the noticeable shift in absorbance peak from ~411 nm
(black line) when there is no amine donor to 330 nm upon immediate
addition of the amine donor (red line), indicating the release of the
internal aldimine to form an external aldimine. After around 30 minutes,
this peak shifts slightly to the left at around ~314 nm indicating the
protonation of the external aldimine to form PMP (blue line). Upon
adding phenylpyruvate, we noticed that the peak at 314 nm persists,
however, at the same time, there is a reformation of a peak in the ~420
nm range (green line). This is indicative of the reformation of an
internal aldimine which is a result of the enzymatic cycle being
complete by transfer of an amine group to phenylpyruvate. The
persistence of the peak at 314 nm can be attributed to the availability
of extra PMP that didn’t transfer an amine group. While these trends
indicate that AATA showed enzymatic activity, it should be noted that
ideally, the reformation of a peak at 411 nm at the end of the cycle
(green line) has a higher absorbance than the peak at ~ 314 nm. The
trends that support the enzymatic activity of AATA are based on a
qualitative analysis of the absorbance and for more conclusive results,
methods such as mass spectrometry should be employed for confirmation.

![](https://static.igem.wiki/teams/4710/wiki/results/results-fig4.jpeg)

Figure 4: UV-VIS Spectrum of three trials of AATA (at 1 mg/mL) in
HEPES buffer bound to PLP through its enzymatic cycle upon addition
of L-phenylalanine (as amine donor) and phenylpyruvate (as amine
acceptor)

### Enzymatic activity of AATA with pyruvate

As seen in [Figure 5](#figure5), similar to the results with
phenylpyruvate, the first three steps of the enzymatic cycle are
correctly represented by peaks at 411 nm (black line), 330 nm (red line)
and 314 nm (blue line) as indication of amine donor interactions with
the transaminase. However, upon addition of the amine acceptor (green
line), there is no change in absorbance indicating that there was no
transfer of amine group. This could also be attributed to factors such
as substrate concentration and assay conditions and therefore
confirmatory tests like mass-spectrometry should be conducted before
concluding the lack of enzymatic activity.

![](https://static.igem.wiki/teams/4710/wiki/results/results-fig5.jpeg)

Figure 5: UV-VIS Spectrum of three trials of AATA (at 1 mg/mL) in
HEPES buffer bound to PLP through its enzymatic cycle upon addition
of L-phenylalanine (as amine donor) and pyruvate (as amine acceptor)

### Enzymatic activity of AATA with 3,5-diiodo-phenylpyruvic acid

Similar to the results with phenylpyruvate, the first three steps of the
enzymatic cycle seen in
[Figure 6](#figure6) are correctly
carried out as represented by peaks at 411 nm (black line), 330 nm (red
line) and 314 nm (blue line) as indication of amine donor interactions
with the transaminase. In the case of our test acceptor, we again see
the reformation of a peak at ~420 nm (green line), similar to
phenylpyruvate. This indicates that the amine group was successfully
transferred and PLP was internally bound again. However, the red shift
of this peak to be further away from the expected peak at ~411 nm could
be caused by reasons other than instrumental error.
**Initial UV-VIS spectroscopy results indicate that AATA has the
potential to accept 3,5,-diiodo-phenylpyruvic acid but this should
be confirmed with more thorough analysis of the product formed.To
make sure that the final product we aimed to synthesize,
3,5-diiodo-L-tyrosine was indeed produced, the assay should be
visualized by mass spectrometry.**

![](https://static.igem.wiki/teams/4710/wiki/results/results-fig6.jpeg)

Figure 6: UV-VIS Spectrum of three trials of AATA (at 1 mg/mL) in
HEPES buffer bound to PLP through its enzymatic cycle upon addition
of L-phenylalanine (as amine donor) and 3,5,-diiodo-phenylpyruvic
acid (as amine acceptor).

### Summary of UV-VIS Results

Our UV-VIS results using AATA across three amine acceptors show some key
inferences.

1. The interaction between AATA and the amine donor,
   L-phenylalanine, is consistent across all trials. This is seen
   in [Figure 7A](#figure7) by the
   decrease in the ratio of absorbance between 411 nm and 330 nm
   upon adding the amine donor. The ratio being above 1 without the
   donor (black bar) and below 1 (red bar) shows the switch in
   absorbance with 411 nm being initially high but upon addition of
   the donor, the absorbance at 330 nm is greater. This is further
   supported by the decrease in absorbance ratio at 330 nm and 314
   nm seen in
   [Figure 7B](#figure7). While the
   decrease in the absorbance ratio doesn’t indicate that the
   absorbance at 330 nm is lesser than 314 nm after waiting 30
   minutes (blue bar) to indicate the formation of PMP, this
   leftward shift in absorbance is more clearly seen in the
   spectrum results in Figures 4,5 and 6. We suspect this to be due to
   a red shift in the spectrum after adding any substrate to the
   reaction mixture. (For instance, all the peaks observed after
   adding any substrates appear at 12 nm more than the expected
   values. These observations are consistent with other experiments
   employing similar methodology.)
2. While there is a shift in absorbance ratios from before and
   after adding phenylpyruvate and 3,5,-diiodo-phenylpyruvic acid
   (test acceptor), no change in absorbance is detected after
   adding pyruvate. To account for the red shift explained above,
   the absorbances at 326 nm and 423 nm (12 nm more than expected
   values) were used to analyze the change in absorbance ratio in
   [Figure 7C](#figure7). From
   [Figure 7C](#figure7), we can
   see that while the change doesn’t cause the absorbance at 423 nm
   to be greater than the absorbance at 326 nm, the decrease in the
   ratio indicates the formation of a peak/ increase in absorbance
   at 423 nm upon addition of the amine acceptor (green bar).

![](https://static.igem.wiki/teams/4710/wiki/results/results-figure7.jpg)

Figure 7: Summary of absorbance data for all three amine acceptors
with AATA (at 1 mg/mL). A: Absorbance ratio at 411 nm/ 330 nm for
AATA alone (black) and AATA immediately after adding an amine donor
(red). B: Absorbance ratio at 330 nm/ 314 nm for AATA immediately
after adding an amine donor (red) and 30 minutes after adding an
amine donor (blue). C: Absorbance ratio at 326 nm/ 423 nm for AATA
30 minutes after adding and amine donor (blue) and after adding the
amine acceptor (green).

A qualitative analysis of the data collected supports the enzymatic
activity of AATA (specifically with phenylpyruvate) and demonstrates
potential for reaction with our test amine acceptor, 3,5,-diiodo-phenyl
pyruvic acid to produce “minithryoxine”. Mass spectrometry would be
needed to confirm the preliminary results presented here.

## Recommendations For Future Studies

---

As mentioned above, in the future, the trends seen in the data above
should be confirmed using mass spectrometry to detect the presence of
the correct intermediates and the final product. Future studies can also
focus on the other methods to characterize successful transamination
reactions. To form replicates of the results obtained in this study,
future studies can also focus on optimizing substrate concentrations to
get clearer results. Changing conditions like pH might also yield better
enzymatic activity. Future studies can also focus on characterizing the
enzymatic activity of HHTA. We were able to indicate the presence of the
active form of HHTA so future studies could focus on different amine
donors that are compatible with reliable UV-VIS readings or try
different methods to characterize successful transamination reactions.
Future studies can also screen for various substrates to understand the
promiscuity of the enzyme.

## Supplementary Data

For any supplementary data regarding the UV-VIS assays, please see the
attached file:

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