iGEM 2022 — Team miamiu

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{% block title %}Notebook{% endblock %}
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## iGEM 2022 Virtual Lab Notebook

## Gel Electrophpresis Results

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{% block title %}Engineering Success{% endblock %}
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# An Overview of MiamiU iGEM Experiments in Context of the Design, Build, Test, Learn Engineering Cycle:

## **I. Design**

SynPhage Specifications: Engineer a phage therapy scaffold that can easily be programmed
to
deliver CRISPR-based genetic circuits targeting bacterial antibiotic resistance and virulence factor
genes.

## A. Phage Scaffold

 M13 has previously been shown to deliver CRISPR-based genetic circuits that kill bacteria based
on the presence of antibiotic resistance genes. However, M13 has limited host range, meaning the
number of bacterial strains M13 can infect. However, T7 and T3 bacteriophages can infect strains of
*E. coli*, including lipopolysaccharide (LPS) and other mutant strains, or related smooth strains, and other enteric microbes. T7 and T3 phages are strictly lytic phages, meaning infects
and lyses bacteria and does not enter the lysogenic life-cycle [[2]](#engineering-citaions "Hess, G. T., Cragnolini, J. J., Popp, M. W., Allen, M. A., Dougan, S. K., Spooner, E., Ploegh, H. L., Belcher, A. M., & Guimaraes, C. P. (2012). M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins. Bioconjugate Chemistry, 23(7), 1478–1487. https://doi.org/10.1021/bc300130z"). Thus, these phages have
attracted strong interest as phage therapies. Moreover, T7 and T3 can readily be programmable to
expand host range to target a wide array of bacterial pathogens, including *E. coli*,
*Yersinia*,
*Klebsiella*, *Salmonella*, and *Shigella*. Therefore, T7 and T3 will be used as
therapeutic phage
scaffolds for delivering CRISPR-based genetic circuits.

## B. Bacterial Host

 We are keen on using *Escherichia coli* as the bacterial host for quantitative and
phenotype
tests. *E. coli* provides the benefits of versatility, ease of handling, a collection of
environmental
phenotypes and applicability within human hosts. Researchers have built a vast number of *E.coli* genomes varying among strains and among techniques, reinforcing the model of
*E.coli*
in the field of genetic engineering or various academic settings [[1]](#engineering-citaions "Lin, T.-Y., Lo, Y.-H., Tseng, P.-W., Chang, S.-F., Lin, Y.-T., & Chen, T.-S. (2012). A T3 and T7 recombinant phage acquires efficient adsorption and a broader host range. PLoS ONE, 7(2). https://doi.org/10.1371/journal.pone.0030954").

As a result of its well
established precedence throughout most scientific disciplines, we would like to use the
*E.coli* as
the cells to exercise the desired gene transformation from the phage. From there we will monitor the
effects of phage infection through growth tests, using optical density (OD) as a proxy, and plaquing
assays.

## C. Phage host range engineering strategy

 Previously, T7 and T3 host range can be altered by swapping out the gene encoding the tail
fiber complex, *gp17*, with tail fiber genes from other phages. However, this strategy is not
straightforward, and requires many iterations to obtain a successful phage with altered host range.
This is because phage tail fibers are very diverse and have many different sizes and structures.
Therefore, we propose engineering T3 and T7 to express a nanobody at the terminus of the tail fiber
complex to alter phage host range.

The nanobody is small in size and possesses the same general
structure. Therefore, engineering phage host range simply requires exchanging out a nanobody that
binds to the target bacterium. Our initial experiments aimed to synthesize a direct fusion of the
nanobody and gp17 tail fiber protein. However, Alphafold2 modeling predicted complex misfolding of
such peptides, regardless of various linker insertions. Therefore, we propose functionalizing the
tail fiber complex post assembly, thus preserving the functionality of both components by letting
them fold prior to functionalization.
 To view the models, visit our [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

## D. Bioconjugation Strategy:

 Sortase A (SrtA) is a transpeptidase, recognizing the sorting signal LPXTG-motif at the
C-terminal domain (CTD) and cleaving at the peptide bond between the Glycine and Threonine residue.
The resulting amino-acyl intermediate is then resolved via nucleophilic attack by a second peptide
containing a hydrophobic poly-glycine fragment at the N-terminal domain (NTD). Sortase catalyzed
reactions are utilized in natural gram-positive bacterial systems to ligate peptides, mostly to
modify bacterial surface proteins to aid in pathogenic attacks against its host (Figure 1) [[3]](#engineering-citaions "Li, J., Zhang, Y., Soubias, O., Khago, D., Chao, F.-an, Li, Y., Shaw, K., & Byrd, R. A. (2020). Optimization of Sortase a ligation for flexible engineering of complex protein systems. Journal of Biological Chemistry, 295(9), 2664–2675. https://doi.org/10.1074/jbc.ra119.012039 "). The
simple addition of short polypeptide tags makes Sortase A useful for modifying target proteins with
a variety of functional and chemical groups (e.g. biotin, fluorophore, etc.) [[4]](#engineering-citaions "Ding, Y., Chen, H., Li, J., Huang, L., Song, G., Li, Z., Hua, X., Gonzalez-Sapienza, G., Hammock, B. D., & Wang, M. (2021). Sortase-mediated phage decoration for analytical applications. Analytical Chemistry, 93(34), 11800–11808. https://doi.org/10.1021/acs.analchem.1c02322").
We utilized the Sortase A pentamutant, an optimized mutant for experimental and ex vivo reactions,
to catalyze the ligation reaction of target protein to phage tail fiber and ultimately produce an
optimal phage vector for delivery of CRISPR-Cas 13 components into a specific bacterial species.
Through analysis of enzyme kinetics, this mutant was found to significantly increase efficiency of
ligation compared to wild type Sortase found in Staphylococcus aureus as shown by higher kcat/Km
value. As a result of these findings, sortase mutant can be found listed as eSrtA in pET29 [[5]](#engineering-citaions "Chen, I., Dorr, B. M., & Liu, D. R. (2011). A general strategy for the evolution of bond-forming enzymes using yeast display. Proceedings of the National Academy of Sciences, 108(28), 11399–11404. https://doi.org/10.1073/pnas.1101046108").

![The mechanism of the Sortase A nicking two ends of peptides based on the sensing of LPXTG and Glycine-rich region.](https://static.igem.wiki/teams/4514/wiki/engineering/image2.jpg)

Figure 1. The mechanism of the Sortase A nicking two ends of peptides based
on the sensing of LPXTG
and Glycine-rich region.

 After choosing the Sortase variant for the mechanical ligation-based modification of the phage
tail
fiber, we needed to design the phage tail and nanobody. Two options, shown below, were the
hypothesized ways to assemble the phage tail and nanobody fusion based on placement of Sortase
recognition sequences.

The first was to encode poly-glycine in the NTD of one peptide and the
Sortase tag (LPXTG motif) on the CTD of the other. When we mix the two types of self-folded peptides
together with the purified Sortase protein followed by our tailless T7 phage (IYPH60), we expect the
assembly of a functional T7 phage with our target protein, properly folded and functional, attached
to its tail fiber.

Figure 2 and Figure 3 depict the various possibilities for arrangement of
nanobody, tail fiber, and linker sequences. Between these options, we concluded that the first would
likely be more successful as compared to the second which would result in the N-terminal domain
being oriented towards the phage head, making it mechanically inaccessible. Therefore, we chose to
design the truncated phage tail fragments to contain the motif LPETGG on the CTD.

![First scheme of the sortase coupling with the motif attached on the CTD of the phage. This is our chosen methodology.](https://static.igem.wiki/teams/4514/wiki/engineering/image4.png)

Figure 2. First scheme of the sortase coupling with the motif attached on
the CTD of the phage. This
is our chosen methodology.

![Second scheme of the soratase coupling with the motif attached on the CTD of the nanobody fragment.](https://static.igem.wiki/teams/4514/wiki/engineering/image1.png)

Figure 3. Second scheme of the soratase coupling with the motif attached on
the CTD of the nanobody
fragment.

## **II. Build, Test, and Learn**

## A. T7 vs T3 phage/ Truncation vs Regular

* Goal:

 Compare the infectivity of both truncated and non-truncated versions of T7 and T3

* Design and build:

 Truncated T7, non-truncated T7, truncated T3 and non-truncated T3 plasmids were constructed
by
Gibson assembly using appropriate primers. (See parts registry)

* Test:

 Plasmids were then transformed into DH5-𝞪 competent cells through heat shock and
transformants
were plated on respective antibiotic selection plates. Engineered phages were then made by using
plasmid containing bacterial strains and infecting with IYP60 (Δgp11, 12, 17) for the assembly
of the different tail fiber constructs.

Plaquing assays were then used as a means of screening
and quantifying the engineered phage species by using respective plasmid containing bacterial
strains. The formation of plaques would suggest the successful transformation, expression of
desired plasmid, and the formation of a number of engineered phages that can be quantified
through counting plaques at the highest serial dilution.

* Learn:

 Plaquing was observed with all phages but truncated T3. Conducted additional plaquing
assays
using the remaining three.

Design and build:

 Phage lysates were acquired from liquid culture based infection of the 3 plasmid containing
bacteria and characterized via plaquing assays. In the plaquing assays, serial dilutions of all
three phage lysates were aliquoted on the following bacterial lawns:

1. Truncated T7 production strain
2. Non-truncated T7 production strain
3. Non-truncated T3 production strain
4. BL21
5. BW25113

Of the three remaining phages, Truncated T7 phage was chosen as the ideal phage for future
experimentation since it had the most robust plaquing results.

## B. Phage tail fiber to nanobody ligation

* Goal:

 The ligation of the nanobody and tail fiber required the preservation of functionality of
both
components.The goal was to achieve a customizable template phage which is capable of having its
infectivity and specificity adapted, according to the target of interest, via sortase A tail
fiber alterations. The proof-of-concept system utilized for this was the nanobody-antigen system
expressed in strains DSG289 (nanobody) and DSG419(antigen).

* Design and build:

 Our initial experiments aimed to synthesize a direct fusion of the nanobody and gp17 tail
fiber
protein by cloning respective genes into a plasmid, using Gibson assembly (see plasmid design
below). This fusion plasmid would lack the Sortase A component. Once constructed, the plasmid
would be transformed into DH5-𝞪 competent cells in order to test for potential functionality or
use as a control in reference to Sortase A mediated reaction.

However, Alphafold2 modeling
predicted complex misfolding of such peptides, regardless of various linker insertions. We
concluded, therefore, that Sortase A coupling would likely be necessary to encourage proper
folding and formation of stable tertiary structures.

* Test:

 Plaquing assay using a serial dilution of T7 Δgp11, 12, and 17 phage lysate, previously
collected
from plaques on T7 transformed production strain, plated on bacterial lawn of T7 tail fiber with
nanobody fusion bacteria.

* Learn and potential design for future research:

 Phage lysate was made through IYP60 amplification in fusion plasmid transformants would
then be
used to test infectivity against DSG419 transformants expressing EPEA tag nanobody protein as
compared to production strain of T7 tail fiber as well as BL21 and BW25113. Similar infectivity
in all strains would suggest unsuccessful attachment and functionality of fusion protein,
indicating the need for the Sortase enzyme catalyzed reaction.

 For future Sortase screening, Sortase and histidine tag components would be cloned into a
plasmid
by a similar procedure. Extraction and purification of the enzyme would follow an affinity
chromatography column protocol. The G-block peptide, which expresses the nanobody with the LPETG
motif, will be connected to a fluorophore which will be used to test activity and efficiency of
the Sortase reaction. Purified Sortase will be used to functionalize the tail fiber with the
nanobody, ex vivo, and the infectivity would then be tested through plaquing assay on DSG419
antigen strain.
 To view the models, visit our [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

## C. Plasmid design

 We designed primers to produce the PCR amplicons encoding the full tail fiber protein, and a
truncated tail fiber protein (the 252 base pairs on the 3' end encoding the 84 C-terminal amino
acids were deleted) for both T7 and T3 bacteriophages. The amplicons were cloned from the plasmids
GEM3RCF(T7) and MGP4185 (T3), respectively. Amplicons were assembled into plasmids by Gibson
Assembly with certain antibiotic resistance genes: Kanamycin for pMGP4185 and Carbapenem for
pGEM3RCF. A more detailed description of our primers and plasmids provided in our [parts registry](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D).

## D. Gibson Assembly

 We followed the protocol provided by BioLabs inc. in the NEBuilder HiFi DNA Assembly Cloning
Kit
Manual [[6]](#engineering-citaions "New England Biolabs. (n.d.). NEBuilder® hifi DNA assembly cloning kit. NEB. Retrieved October 11, 2022, from https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Protocols,%20Manuals%20&%20Usage_Manuals").

## E. Cas pathway

 The continued development of CRISPR-Cas systems have provided us with numerous ways to tackle
problematic aspects of microbes, including antibiotic resistance. CRISPR-Cas systems offer the
ability to specifically target genes of interest for degradation. Well-characterized systems to
achieve this include those involving Cas9 or Cas3.[[7]](#engineering-citaions "Xu, Y., & Li, Z. (2020). CRISPR-Cas Systems: Overview, innovations and applications in human disease research and Gene Therapy. Computational and Structural Biotechnology Journal, 18, 2401–2415. https://doi.org/10.1016/j.csbj.2020.08.031")

 CRISPR-Cas components can be delivered through a phage vector and target antibiotic resistance
and
virulence factor genes, which can either be located on the bacterial chromosomal or plasmid. We
think Cas13a, a type VI class II CRISPR can be quite promising. Cas13 exhibits promiscuous RNA
cleavage activity which is highly detrimental to a microbial system when sequence complementarity is
accomplished.

[8]

This allows for post-transcriptional interference with the gene of interest (antibiotic resistance
genes, pathogenicity genes, virulence factor genes etc.) and the other non-specific bacterial RNA as
well [[9]](#engineering-citaions "Kiga, K., Tan, X.-E., Ibarra-Chávez, R., Watanabe, S., Aiba, Y., Sato’o, Y., Li, F.-Y., Sasahara, T., Cui, B., Kawauchi, M., Boonsiri, T., Thitiananpakorn, K., Taki, Y., Azam, A. H., Suzuki, M., Penadés, J. R., & Cui, L. (2020). Development of CRISPR-CAS13A-based antimicrobials capable of sequence-specific killing of target bacteria. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-16731-6"). Additionally, this allows for a level of selection towards bacteria expressing
pathogenicity or AMR genes (disease/problem-causing genes) while essentially harmless bacteria are
able to escape RNA damage due to lack of expression of pathogenic genes regardless of their presence
in their genome, making it an eduactive component to be packages into our engineered phages.
Similar to Cas9, the Cas13a system utilizes a guide RNA; after the gRNA binds to the target RNA, the
Cas13a would cleave both the target and other non-specific ssRNA as well as the region is in close
distance to the ribonucleoprotein complex (figure 4) [[10]](#engineering-citaions "Burmistrz, M., Krakowski, K., & Krawczyk-Balska, A. (2020). RNA-targeting CRISPR–CAS systems and their applications. International Journal of Molecular Sciences, 21(3), 1122. https://doi.org/10.3390/ijms21031122").

![Schematic representation of Cas13a broad cleavage activity on RNA sequences.](https://static.igem.wiki/teams/4514/wiki/engineering/image3.png)

Figure 4. Schematic representation of Cas13a broad cleavage activity on RNA
sequences.

## F. General procedure

* Design:

 After determination of target genes, plasmids and respective primers are designed through
Benchling software. Amplicon size and primer melting temperature noted for proper adjustment of
procedures.

* Build and test:

1. Gene of interest is amplified through PCR using appropriate plasmid template and primers.
2. PCR products (amplicons) are characterized by agarose gel electrophoresis through examining
   the base pair length.
3. Confirmed amplicons are extracted from the gel and the DNA is eluted from the column by
   using the Qiagen Gel Extraction Kit.
4. The amplicon product concentration is measured by UV/Vis absorption at 260 nm wavelength via
   a Nanodrop Spectrophotometer.
5. Once amplicon concentrations are known, the Gibson assembly is run to make the plasmid of
   interest.
6. The constructed plasmid is transformed into competent cells by heat shock and plated on
   appropriate antibiotic selection plates.
7. Upon successful growth of transformants, colonies are used to make an overnight liquid
   culture.
8. Overnight culture is used to make a freezer stock, amplify the phage of interest, and also
   for plaquing assay to synthesize characterize the amplified phage sample.
9. A plasmid extraction from the transformed colonies will follow subsequent to successful
   plaquing and quantitative PCR for characterization of the plasmid.
   Quantifying plasmid
   and amplicon concentrations:
   The concentrations of plasmid and/or PCR amplicons were
   quantified with the NanoDrop Spectrophotometer. DNA quantification is essential to Gibson
   assembly procedures.

* Learn:

 The modeling of the nanobody and tail fiber fusion revealed that there will not be proper
folding of the nanobody. As a result, this will be used as a control when constructed. The
improper folding suggests improper or malfunction of the nanobody leaving the phage fusion as
unable to infect the DSG419 (strain expressing nanobody antigen).

 Negligible detection of the transformed colonies on selective antibiotic agar plates can
potentially indicate toxicity of engineered plasmids or the exhaustion of cellular machinery.
Other obstacles we faced included difficulty with PCR amplification of large amplicons as well
as the low efficiency of 3-piece Gibson assembly.

 Because we set out to make a therapeutic for human systems, we were faced with
inherently complicated design considerations. These included promoting robust silencing
of antimicrobial resistance genes while avoiding disruption of the commensal gut microbiome. In
addition, we sought to incorporate emerging synthetic biology techniques such as sortase
mediated protein engineering and nanobody recognition. Extensive trial and error is still
required, therefore, before all aspects of our desired system are fully optimized.

## References

1. Citorik, R. J., Mimee, M., & Lu, T. K. (2014). Sequence-specific antimicrobials using
   efficiently delivered RNA-guided nucleases. Nature Biotechnology, 32(11), 1141-1145.
<https://doi.org/10.1038/nbt.3011>2. Krüger, D. H., & Schroeder, C. (1981). Bacteriophage T3 and bacteriophage T7 virus-host cell interactions. Microbiological Reviews, 45(1), 9–51.
<https://doi.org/10.1128/mr.45.1.9-51.1981>3. Yehl, K., Lemire, S., Yang, A. C., Ando, H., Mimee, M., Torres, M. D., de la Fuente-Nunez, C., & Lu, T. K. (2019). Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis. Cell, 179(2).
<https://doi.org/10.1016/j.cell.2019.09.015>4. Yosef, I., Goren, M. G., Globus, R., Molshanski-Mor, S., & Qimron, U. (2017). Extending the host range of bacteriophage particles for DNA transduction. Molecular Cell, 66(5).
<https://doi.org/10.1016/j.molcel.2017.04.025>5. Blount, Z. D. (2015). The unexhausted potential of E. Coli. ELife, 4.
<https://doi.org/10.7554/elife.05826>6. Li, J., Zhang, Y., Soubias, O., Khago, D., Chao, F.-an, Li, Y., Shaw, K., & Byrd, R. A. (2020).
   Optimization of Sortase a ligation for flexible engineering of complex protein systems. Journal
   of Biological Chemistry, 295(9), 2664-2675.
<https://doi.org/10.1074/jbc.ra119.012039>7. Ding, Y., Chen, H., Li, J., Huang, L., Song, G., Li, Z., Hua, X., Gonzalez-Sapienza, G.,
   Hammock, B. D., & Wang, M. (2021). Sortase-mediated phage decoration for analytical
   applications. Analytical Chemistry, 93(34), 11800-11808.
<https://doi.org/10.1021/acs.analchem.1c02322>8. Chen, I., Dorr, B. M., & Liu, D. R. (2011). A general strategy for the evolution of bond-forming
   enzymes using yeast display. Proceedings of the National Academy of Sciences, 108(28),
   11399-11404.
<https://doi.org/10.1073/pnas.1101046108>9. New England Biolabs. (n.d.). NEBuilder® hifi DNA assembly cloning kit. NEB. Retrieved October
   11, 2022, from
<https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Protocols,%20Manuals%20&%20Usage_Manuals>10. Xu, Y., & Li, Z. (2020). CRISPR-Cas Systems: Overview, innovations and applications in human
    disease research and Gene Therapy. Computational and Structural Biotechnology Journal, 18,
    2401-2415.
<https://doi.org/10.1016/j.csbj.2020.08.031>11. Kiga, K., Tan, X.-E., Ibarra-Chávez, R., Watanabe, S., Aiba, Y., Sato'o, Y., Li, F.-Y.,
    Sasahara, T., Cui, B., Kawauchi, M., Boonsiri, T., Thitiananpakorn, K., Taki, Y., Azam, A. H.,
    Suzuki, M., Penadés, J. R., & Cui, L. (2020). Development of CRISPR-CAS13A-based antimicrobials
    capable of sequence-specific killing of target bacteria. Nature Communications, 11(1).
<https://doi.org/10.1038/s41467-020-16731-6>12. Burmistrz, M., Krakowski, K., & Krawczyk-Balska, A. (2020). RNA-targeting CRISPR-CAS systems and
    their applications. International Journal of Molecular Sciences, 21(3), 1122.
[https://doi.org/10.3390/ijms21031122](https://doi.org/10.3390/ijms21031122%20)

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