iGEM 2023 — Team duke

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# Notebook

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# Engineering

[Secretion Study](#secretion-study)
[Selecting tag-cargo combinations](#tag-cargo)
[Iterative Design of Secretion Schema](#iterative-design)
[Exploring combined effect of promoter strength and signal peptide
type](#prom-strength)

## Secretion Study

The amplification of the T-cell activation response in our genetic
circuit relies on secretion of target antigen ecto-domains into the
extracellular space. Endogeneously, only some proteins are secreted
from cells, while others operate inside of the cell where they are
created. When engineering proteins that need to be secreted, it is
neccesary to "tag" them via the addition of N-terminal peptides. These
secretion tags are sequences of 15-20 amino acids that signal to the
cell that the tagged protein needs to be sent outside of the cell.

In the genetic engineering space, these fused tags are known to affect
total expression of the linked protein and downstream secretion
efficiency, although this has not been measured in a high-throughput
or rigorous fashion in mammalian synthetic biology systems.**As part of our work on ICARUS, we aimed to build and test a catalog
of secretion schema for mammalian synthetic biology which identifies
the most effective secretion tag and promoter strength for a given
size of cargo.**

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-secretion-schema.png)

## Selecting tag-cargo combinations

Because the ecto-domains of specific cancer antigens can vary in size,
we wanted to determine the best secretion tag to use for a wide range
of cargo sizes. We selected four proteins ranging from 184 to 1024
amino acids to use as test cargo:

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-cargos.png)

We then identified commonly used secretion tags for mammalian cells
(Reference: Commonly used leader peptide sequences for mammalian cells
expression, NovoPro) and selected four tags that endogenously signal
the secretion of varying-sized proteins, along with a control.

## Iterative Design of Secretion Schema

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-eng-cycle.png)

We went through three design cycles to determine how to best construct
our secretion catalog. In the end, we found the most success with the
cargo-swap protocol, a method we developed and used to successfully
build and test secretion of our four different-sized cargos in
combination with the five unique secretion tags.

### 1. Site-directed mutagenesis

Design

Typically, in order to combine gene fragments, it is neccesary to
clone each fragment separately then attach them using Gibson
assembly. However, because the secretion tags we wanted to attach
to the cargo were small (40-70bp), we first tried to use a
technique called site-directed mutagenesis (SDM). SDM allows for
the insertion of small gene fragments into double-stranded plasmid
DNA via PCR, bypassing the need for Gibson assembly. With SDM, the
genetic sequences for the insert is tacked on to the ends of the
forward and reverse primers that will be used to copy the
backbone. Then, when the primers attach to the backbone during
PCR, the insert becomes integrated into the backbone. The end
result is numerous copies of the backbone, with the insert added
in its proper location.

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-sdm.png)

Build

To construct plasmids via site-directed mutagenesis, we designed
and ordered primers that targeted the insertion site and contained
the gene for the secretion tag. We used our previously-constructed
pcDNA5-sfGFP backbone for these proof-of-concept experiments,
exchanging only the secretion tag across the different trials. We
ran multiple trials of PCR with the backbone and specific primers,
then ran gel electrophoresis to determine if amplification
occured. Then we transformed the plasmids into E.coli and let them
grow up before isolating the DNA and sending it for sequencing to
determine if the secretion tag had been properly inserted.

See "Site-Directed Mutagenesis Protocol" under "Experiments" for
further detail.

Test

Because the secretion tags are very short sequences, it is
impossible to conclude from gel electrophoresis alone whether or
not the tag was properly inserted via PCR. As a result, we had to
carry out the entire transformation, culture, and DNA isolation
protocol before obtaining sequencing results. Despite several
attempts with each secretion tag, upon receiving the sequencing
results, only one of the four peptides, HSA, had been properly
inserted.

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-sdm-seq.png)

We then successfully transfected the HSA-sfGFP construct into HEK
cells and measured secretion of sfGFP, which served as a valuable
proof-of-concept (See Results > HSA for further details). However,
the overall success rate of site-directed mutagenesis was low for
this experiment.

Learn

Through our experiments in site-directed mutagenesis, we learned
that using small inserts attached to the primers themselves is not
always reliable. Colonies were grown, but the plasmids did not
contain the insert. Some possible ways to troubleshoot the
experiments could be redesigning the primers, increasing the time
of the DpnI digest to decrease the likelihood that we transform
with an unmodified plasmid, or picking multiple colonies for
inoculation into liquid culture to obtain more sets of DNA that
could contain the proper insert.

### 2. Two-fragment Gibson assembly

Design

Because we were unable to insert the secretion tags as part of the
primers using site-directed mutagenesis, our next idea was to
order short fragments containing only the secretion tags. We
planned to run PCR on the secretion tags as well as the
cargo-containing backbone pieces, then use Gibson Assembly to
combine the backbone and the insert. This involves more steps than
site-directed mutagenesis, but would possibly be more reliable
since we'd already had experience with Gibson Assembly.

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-2frag-gibson.png)

Build

We ordered five short DNA fragments, each containing one of the
secretion tags (IFNa, IGK8, CTR, SP, and HA). We planned to insert
the tags into the pcDNA5-sfGFP backbone we'd constructed, using
sfGFP as our test cargo for these experiments. We multiplied the
DNA using PCR, ran gel electrophoresis to confirm that we had
fragments of the proper sizes, and then attempted to do Gibson
Assembly on the fragments. Again, we had to transform the
assembled plasmids into E.coli, plate the cells, inoculate into
liquid culture, then extract and purify the DNA before we could
obtain sequencing results.

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-inserts-bb.png)

Test

After sending the DNA to get sequenced, we found that none of the
Gibson Assembly rounds had been effective. Most of the results we
had showed the pcDNA5-sfGFP plasmid unmodified, inicating that the
small inserts containing the secretion tags had not been inserted.

Learn

From these experiments, we learned that Gibson Assembly with very
small fragments is difficult. With an insert of only ~60 base
pairs, it is likely that the T5 exonuclease enyzyme used for
Gibson Assembly digested the fragments before they could be
attached. We needed to find a way to attach the secretion tags to
the cargo and backbone that didn't involve Gibson Assembly of such
small parts.

### 3. Cargo-swap protocol

Design

At this point, we had realized that we could not count on being
able to insert or attach the small signal peptides into the larger
cargo-backbone constructs using any traditional insertion method.
As an alternative, we decided to order longer sequences as gBlocks
that contained a signal peptide already attached to one of our
cargos. This meant that when we performed Gibson Assembly with the
pcDNA5 backbone, we would end up with five completed plasmids,
each containing a different cargo with a different secretion tag
already attached.

Once we had these plasmids complete, it would theoretically be
easier to swap out the cargo sequences and leave the secretion
tags attached to the backbones. While the secretion tags are only
around 20 amino acids, the cargo molecules are much larger,
between 184-1024 amino acids. This would mean that it would be far
easier to cut out and duplicate these larger genes, then perform
Gibson Assembly to attach them into the secretion tag-backbone
constructs.

![](https://static.igem.wiki/teams/4646/wiki/engineering/igem-cargo-swap.png)

Build

Constructing the entire catalog of tag-cargo permuations involved
many rounds of PCR and Gibson Assembly. Each of the five tag-cargo
sequences we ordered had to be inserted into the pcDNA5 backbone.
Once this was completed, primers were needed to cut out both the
cargo and the tag-backbone fragment from each plasmid. This
resulted in a full set of each cargo as well as each tag attached
to the backbone. Finally, each cargo could be added to the
different tag-backbone fragments. Following Gibson Assembly, all
plasmids were transformed into E.coli, for culturing, then the DNA
was extracted and sequenced.

Test

With this method of swapping out just the cargo from
pre-constructed complete plasmids, we finally saw large-scale
success in the construction of our secretion catalog.

Learn

Gibson Assembly with the larger cargo fragments was far easier
than with the small secretion tag fragments. Despite some
challenges with the PCR of the original parts, we were able to
successfuly build the plasmids for our secretion catalog.

## Exploring combined effect of promoter strength and signal peptide type

Design

In this study, we collaborated with Asimov to test the combined
effect of promoter strength and signal peptide type on the secretion
of proteins using a flow cytometry assay. Commonly used promoters
such as CMV (BBa\_I712004), CAG (BBa\_K2217010), EF1$\alpha$
(BBa\_K2520023), and SV40 (BBa\_K1722006) were selected for the Asimov
flow cytometry scaffold. Additionally, we selected 5 signal peptides
previously used to secrete proteins in mammalian systems as our
candidates for the antigen signal peptide.

As part of our experimental design, we also included the HA epitope
tag as our negative secretion control. This control group serves as
a baseline reference, enabling us to normalize the secretion levels
of the proteins against those with the HA-tag, and make meaningful
conclusions about the impact of the different variables being
tested. We assume that HA-tagged proteins will not secrete through
normal pathways, but that death and lysis of cells may result in
background levels of fluorescence during experiments.

![](https://static.igem.wiki/teams/4646/wiki/duke-asimov-designs.png)

The Asimov scaffold expresses two reporter proteins, mEYFP as the
reporter of secretion and TagBFP as the reporter of transfection
efficiency. The expression of these two reporters allowed us to
normalize the secretion results across different transfection
efficiencies and make meaningful conclusions about the secretion
efficiency, considering that TagBFP has no tags for secretion and a
common MmPgk1 promoter in all constructs. By analyzing the mEYFP
median fluorescence intensity (MFI) normalized to the TagBFP MFI, we
can compare how signal peptides reduce intracellular mEYFP as a
quantitative measure of secretion.

Build

The plasmid DNA was synthesized by GeneScript. The scaffolds
containing CAG promoter were not constructed due to synthesis issues
(please see our parts page for the full list of tested constructs).

Test

HEK293 Freestyle cells were transfected with 300 ng of DNA into 1
000 000 cells per mL in a 170 µL total culture volume in 96-well
plates. Following transfection, the cells were incubated for 48
hours in an incubator at 350 rpm, 37°C, 8% CO$\_2$. The cells were
then resuspended in 200 µL of PBS and analyzed on the flow cytometer
(please see our measurement page for results of this study).

Learn

Flow cytometry data analysis indicated that the promoter strength
plays a major role in the amount of the protein secreted as
indicated by HA-tagged constructs, with EF1⍺ and CMV promoters
having the highest abundance. The signal peptide type did influence
this metric in a significant manner, as indicated by the significant
reduction in intracellular mEYFP for all constructs. Perhaps, the
selection of our signal peptides was not broad enough to reveal the
most optimal candidate as these appear to have similar YFP/BFP
ratios. This insight will aid our promoter and signal peptide
selection choices for the future iteration of the antigen secretion
construct.

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# Experiments

## Wet Lab Protocols

In conducting our experiments this year we used the following protocols:

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# Results

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