iGEM 2022 — Team mit-mahe


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{% block title %}Engineering Success{% endblock %}
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# Designing the peptide

**The Design–Build–Test–Learn (DBTL) cycle** is integral to any synthetic biology-based
engineered solution for biological problems. As a result, to arrive at the best possible peptide, we performed
**multiple iterations** of the engineering cycle. Each iteration involved designing our peptide, building it and
finally testing it *in-silico*. The changes involved in each iteration and the information learnt have been
documented.

![The design, build test learn cycle graphic](https://static.igem.wiki/teams/4200/wiki/engineeringsuccess/dbtlcycle-compressed-25.jpeg)

## The DBTL cycle *Design, Build, Test and Learn*

The predicted structures of MAM7 and Heparin Binding Domain (HBD) of Fibronectin were
docked with each other on ClusPro [1]-[4], followed by visualisation and observation of these interacting residues
on PyMOL [5]. The sequence signature of the structural repeats in the proteins of Fibronectin and MAM7 were detected
using HHrepID [6],[7]—a tool for *de novo* identification of repeats in protein sequences. This confirmed the
existence of 5 continuous repeats of type 1 modules, forming the **Heparin-Binding Domain** of Fibronectin and
the presence of seven Mammalian Cell Entry domains (MCEs) in MAM7. Fibronectin is known to require **five tandem
MCE domains** to dock with MAM7; as a result, designing a peptide that would bind to one of the central MCE
domains was deemed sufficient [8].

Each of the five repeats of the type I module present in Fibronectin was considered an
individual peptide, and the structure for each of these peptides was predicted. Amber, an energy mininmization tool,
was employed to stabilize the predicted peptide structures. The peptides were then docked with MAM7 to test its
binding using ClusPro [1]-[4]. It was observed that the peptides numbered three and five derived from modules three
and five of Fibronectin, respectively, docked with the MCE3 of MAM7, gave the highest docking scores.

It was seen that the **fifth peptide** had the highest docking score with MCE3. Thus,
this peptide was taken as the basis for the **next set of modifications**.

 ![Fibronectin docked with MAM7](https://static.igem.wiki/teams/4200/wiki/multiplepeptide5.png)

Fig 1 :Fibronectin docked with MAM7(viewed on PyMOL[1]-[4])

**Schrodinger Maestro**[9] was used to visualise and study the specific amino acids
involved in the interaction between the peptide and MAM7. The residues within 5 Å were assumed to be close enough
for the interaction and thus were considered during the evaluation. Further, this was verified by subjecting the
peptide to an alanine scan on BUDE. An alanine scan replaces all the amino acid residues in a peptide with alanine
one at a time and then compares the values of the respective Gibbs free energies to determine the extent of
contribution of every amino acid in the docking process [10]. **Five** such mutatable positions were identified
in the wild type. This was followed by improving the interactions by substituting aforementioned amino acids. These
replacements were made based on the study [11], but each of them had a plethora of possibilities. For example,
Methionine (present at the 29th position) could be replaced by Glutamic acid, Aspartic acid, Phenylalanine,
Tyrosine, Tryptophan, Histidine or Leucine.

Therefore, the docked structure was subjected to **mutation Cutoff Scanning Matrix**
(mCSM) [12], which helped us analyse which mutations were truly beneficial. With newly **learnt** information,
the peptide was subjected to **new changes**. Hence, it was necessary to predict the structure of the peptide and
dock it with MAM7 at every stage. The docked complex was subjected to an alanine scan to account for conformational
changes by the substitutions, that mCSM does not account for, and to verify the interactions. This gave us twelve
variants of the peptide with different combinations of the various substitutions that could be made. These were then
visualised on Schrodinger Maestro and further analysed using **Prodigy** to identify the strongly interacting
peptides.

**Four** of the **twelve** peptides showed an increased binding affinity, and the
best among these was selected for the next round. The mutations performed in the previous iteration introduced new
amino acids in the docked structure, opening up an avenue for a new set of substitutions and hence the process was
repeated. Subsequently, a new set of variants were obtained (16 variants). These were further subjected to
**docking** and analysis to select the next best peptide. At this stage, to prevent the peptides from mutually
interacting and consequently decreasing their interaction efficiency with MAM7, we performed **AGGRESCAN** [13]
on the four peptides obtained from the last iteration, to highlight the **hotspots of aggregation**. One such
hotspot involving **eight** amino acids was observed and was rectified by substituting **Cytosine** at the
34th position with **Asparagine** in every peptide. After this process, our very own
software—**GRASP**, helped us increase the scope of possible mutations.

Two rounds of running the peptide through the software gave us the final one. The
structure of the peptide was then predicted and docked with MAM7, and run through Prodigy [14],[15] to compare its
binding affinity and Kd value with the previous variants. These values are tabulated below:

**Table- Peptide variants with gibbs free energy and dissociation constant**
![Table file with peptide variant vs energy](https://static.igem.wiki/teams/4200/wiki/engineeringsuccess/ddg-table-finally.png)

**References:**

1. I. T. Desta, K. A. Porter, B. Xia, D. Kozakov, and S. Vajda, “Performance and Its Limits in Rigid Body
   Protein-Protein Docking,” Structure, vol. 28, no. 9, pp. 1071-1081.e3, Sep. 2020, doi: 10.1016/j.str.2020.06.006.
2. S. Vajda et al., “New Additions to the ClusPro Server Motivated by CAPRI”, doi: 10.1002/prot.25219.
3. D. Kozakov et al., “The ClusPro web server for protein-protein docking”, doi: 10.1038/nprot.2016.169.
4. D. Kozakov et al., “How Good is Automated Protein Docking?”, doi: 10.1002/prot.24403.
5. PyMOL The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC.
6. L. Zimmermann et al., “A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its
   Core,” J. Mol. Biol., vol. 430, no. 15, pp. 2237–2243, Jul. 2018, doi: 10.1016/j.jmb.2017.12.007.
7. F. Gabler et al., “Protein Sequence Analysis Using the MPI Bioinformatics Toolkit,” Curr. Protoc. Bioinforma.,
   vol. 72, no. 1, Dec. 2020, doi: 10.1002/cpbi.108.
8. A. M. Krachler and K. Orth, “Functional characterization of the interaction between bacterial adhesin
   Multivalent Adhesion Molecule 7 (MAM7) protein and its host cell ligands,” J. Biol. Chem., vol. 286, no. 45, pp.
   38939–38947, Nov. 2011, doi: 10.1074/JBC.M111.291377.
9. Maestro Schrödinger Release 2022-3:Maestro, Schrödinger, LLC, New York, NY, 2021.
10. A. A. Ibarra et al., “Predicting and Experimentally Validating Hot-Spot Residues at Protein-Protein Interfaces,”
    ACS Chem. Biol., vol. 14, no. 10, pp. 2252–2263, Oct. 2019, doi:
    10.1021/ACSCHEMBIO.9B00560/SUPPL\_FILE/CB9B00560\_SI\_002.XLSX.
11. N. Z. Xie, Q. S. Du, J. X. Li, and R. B. Huang, “Exploring Strong Interactions in Proteins with Quantum
    Chemistry and Examples of Their Applications in Drug Design,” PLoS One, vol. 10, no. 9, p. 137113, Sep. 2015, doi:
    10.1371/JOURNAL.PONE.0137113.
12. D. E. V. Pires, D. B. Ascher, and T. L. Blundell, “mCSM: predicting the effects of mutations in proteins using
    graph-based signatures,” Bioinformatics, vol. 30, no. 3, pp. 335–342, Feb. 2014, doi:
    10.1093/BIOINFORMATICS/BTT691.
13. O. Conchillo-Solé, N. S. de Groot, F. X. Avilés, J. Vendrell, X. Daura, and S. Ventura, “AGGRESCAN: A server for
    the prediction and evaluation of ‘hot spots’ of aggregation in polypeptides,” BMC Bioinformatics, vol. 8, no. 1,
    pp. 1–17, Feb. 2007, doi: 10.1186/1471-2105-8-65/FIGURES/1.
14. A. Vangone and A. M. Bonvin, “Contacts-based prediction of binding affinity in protein–protein complexes,”
    Elife, vol. 4, Jul. 2015, doi: 10.7554/eLife.07454.
15. L. C. Xue, J. P. Rodrigues, P. L. Kastritis, A. M. Bonvin, and A. Vangone, “PRODIGY: a web server for
    predicting the binding affinity of protein–protein complexes,” Bioinformatics, p. btw514, Aug. 2016, doi:
    10.1093/bioinformatics/btw514.

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# Cloning and Expression System

For our experimentation, expression of MAM7 was done by using our chassis– *E.coli*
BL21 and the vector pET-22b(+).

![MAM7 cloning](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/mam7-cloning-plasmid.png)

*Fig 1: MAM7 cloning and expression vector system* [1]

With our progress in the project, our Wet Lab geared up on a detailed itinerary of planned
experiments. This
curated
handbook includes various experiments like plasmid isolation, competent cell preparation, Ni-NTA column
chromatography, etc. The primary objective is to help other iGEM teams carry out these recombinant experiments and
provide them with the opportunity to improve their current experimental designs.

With our progress in the project, our Wet Lab geared up on a detailed itinerary of
experiments ready to go ahead
with. Unfortunately due to lack of time we could not execute the lab work as planned. The [Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D) page consists of our
experimentation handbook and a lab bulletin that summarises our journey so far in the lab. We curated a handbook
such that anyone could refer to it in order to gain a better understanding of our experimentation itinerary. In case
anyone happens to pick a project in the same trajectory as ours, this could serve as a guide for them. This handbook
has a variety of experiments that we have performed and plan on performing further like—plasmid isolation, peptide
purification. Despite our iGEM season coming to an end soon enough, we intend to try to continue our
experimentation.

# Experimentation Timeline

Here's our week wise experimentation timeline!

Week 0
  Week 1
  Week 2
  Week 3
  Week 4
  Week 5
  Week 6
  Week 7

### Week 0

We visited the Enteric Disease Division at KMC, Manipal and observed the
protocols for culturing, storage and detection of *Vibrio parahaemolyticus* strains under
supervision.

![Vibrio parahaemolyticus blood agar plate culture](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-0-picture.jpg)

*Vibrio parahaemolyticus* blood agar plate
culture

### Week 1

* Primary Sub-culturing of *E. coli* DH5α.
* Quantifying growth of Primary culture of DH5α

**Result**
 OD600 = 2.5

* Gram staining was performed for the first subculture.

**Result**
 *E. coli* DH5α cells were
observed to be pink.
 Cell debris were observed.

**Troubleshoot**
 Initial smearing
could be done more gently.

* Secondary subculture was performed
* Quantifying growth of secondary subculture of the culture

**Result**
 OD600 = 1.4

* Gram staining was performed for the secondary subculture

**Result**
 *E. coli* DH5α cells were
observed to be pink.
 Some crystal violet stain residues were observed

 Many cells were clumped together

 Lesser cell debris were observed

**Troubleshoot**
 Washing with water
can be done better and longer.

* Multiple subcultures were prepared, and *E. Coli* DH5α growth was confirmed using
  Indole test and Gram staining

![E.coli DH5-alpha gram staining observation](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-1-gram-staining.jpg)

*E. coli* DH5α gram staining observation

### Week 2

* Primary culturing of pET-22b(+) transformed *E. coli* DH5α from prepared glycerol stock.
* Quantification of transformed *E. coli* culture.

**Result**
 OD600 = 3

* Secondary culturing of *E. coli* DH5α.
* Quantification of transformed *E. coli*

**Result**
 OD600 = 0.7

* Plasmid isolation of pET-22b(+) from transformed *E. coli* (Trial 1)
* Quantification of Plasmid DNA.

**Result**
 DNA concentration was found to
be 38.1 ng/μL.

* Gel electrophoresis to confirm the presence of isolated plasmid.

**Result**
 - The plasmid was not observed.

 - No RNA contamination was observed.

**Troubleshoot**
 - The culture was
advised to be kept until an OD600value of 1-1.2 was obtained.
 - The concentration of
plasmid should be around 100 ng/μL.

![pET-22b+ plasmid Isolation](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-2-isolated-plasmid.jpg)

pET-22b(+) plasmid Isolation:
 No vector bands
were observed

### Week 3

* Primary subculturing of pET-22b(+) transformed *E. coli* DH5α from prepared transformed
  plates.
* Quantification of transformed *E. coli* culture

**Result**
 DH5α

 -> Culture 1 OD600 = 0.4

 -> Culture 2 OD600 = 0.6

* Secondary overnight sub-culturing of pET-22b(+) transformed *E. coli*
* Plasmid isolation of pET-22b(+) from transformed *E. coli* (Trial 2)
* Quantification of Isolated plasmid.

**Result**
 DNA concentration was found to
be 69 ng/μL.

* Gel electrophoresis for Isolated Plasmid

**Result**
 The plasmid was successfully
isolated.

![pET-22b+ plasmid isolation.](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-3-isolated-plasmid.jpg)

pET-22b(+) plasmid isolation.
 Vector bands were
observed in well number 4 and 5

### Week 4

* Primary Subculturing of pET-22b(+) transformed *E. coli* DH5α
* Quantification of Transformed *E. coli*

**Result**
 Glycerol stock culture
OD600 = 2.1

 Plate culture OD600not measurable

* Plasmid isolation and checking sample concentrations
* Checking plasmid DNA concentration using nanocuvette

**Result**
 The concentration of DNA
isolated from plate culture was found to be 48.2 ng/μL.
 The concentration of DNA isolated from
stock culture was found to be 18.3 ng/μL.

* Gel electrophoresis for isolated plasmid samples

**Result**
 The plasmid was successfully
isolated.

![pET-22b+ plasmid isolation.](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-4-isolated-plasmid.jpg)

pET-22b(+) plasmid isolation.
 Vector bands were
observed in well number 4 and 5

### Week 5

* Single digestion of pET-22b(+). Was performed to check the efficiency of our enzymes. Reaction
  1
  was performed using NdeI enzyme. Reaction 2 was performed using the XhoI enzyme.
* Gel electrophoresis of single Digested pET-22b(+)

**Result**
 The digested band was in the
5.5kb range according to the DNA ladder, while the plasmid itself is
5493bp. Thus, single digestion of pET-22b(+) using NdeI and XhoI was successful.

* Double digestion of pET-22b(+)
* Gel electrophoresis of double digested pET-22b(+) samples
* Primary Subculturing of pET-22b(+) transformed *E. coli* DH5α
* Quantification of Transformed *E. coli*

**Result**
 - Plate culture 1:
OD600 = 0.2

 - Plate Culture 2: OD600 = 0.6

* Plasmid Isolation of pET-22b(+) from *E. coli* DH5α
* Concentrations of Isolated plasmid

**Result**
 The concentration of DNA from
plate culture 1 was found to be 54.4 ng/μL.
 The concentration of DNA from plate culture 2 was
found to be 48.8 ng/μL.

* Gel electrophoresis of isolated plasmid samples
* Dissolving lyophilized IDT MAM7 gene fragment

**Result**
 The concentration of isolated
plasmid was found to be 11.3 ng/μL.

* PCR Amplification of MAM7
* Running of gel electrophoresis of amplified MAM7
* Purification of MAM7 amplicon using PCR cleanup kit

**Result**
 After purification, the
concentration of purified MAM7 was way too low.

* pET-22b(+) Plasmid Isolation using Miniprep Kit

**Result**
 Plasmid DNA concentration from
plate culture 1 was found to be 55.1 ng/μL.
 Plasmid DNA concentration from plate culture 2 was
found to be 56.9 ng/μL.

![Double Digestion of pET22-B+.](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-5-double-digest.jpg)

Double Digestion of pET-22b(+)
 The digested
vector was observed In well number 6

![pET22-b+ plasmid isolation](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-5-isolated-plasmid.jpg)

pET-22b(+) plasmid isolation

 Vector bands were observed in well number 4 and 5

![PCR Amplification of MAM7](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-5-mam7-pcr.jpg)

PCR Amplification of MAM7

 The amplified MAM7 gene was observed in well 2

![Single digestion of pET22-B+.](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-5-single-digest.jpg)

Single digestion of pET-22b(+)
 The digested
vector bands were observed in well number 4 and 5

### Week 6

* PCR Amplification of the MAM7 gene
* Dissolving lyophilized IDT MAM7 gene fragment

**Result**
 OD600 = 1.3 ng/μL.

* MAM7 Primer Synthesis for AMplification of gene fragment
* PCR Reaction Mix Prep for MAM7 gene amplification
* PCR Gel Purification of Amplified MAM7
* pET-22b(+) and MAM7 Ligation
* Chemical Preparation for PEG Method
* Competent cell preparation using the PEG method

**Result**
 The molar concentration of
glucose and Mg2+ was rectified for the TSS preparation
needed
for
competent cell preparation and transformation.

* Transformation of pET-22b(+) was performed via PEG Method (Trial 1)

**Result**
 We determined the
transformation efficiency and we obtained the highest number of isolated
colonies. Hence, it was successful.

![PCR Amplification of MAM7.](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-6-mam7-pcr.jpg)

PCR Amplification of MAM7.
 The amplified MAM7
was observed in well 2, 3 and 4

![1st Ligation of MAM7 insert and pET22-b+ vector](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-6-first-ligation.jpg)

1st Ligation of MAM7 insert and pET22-b+ vector

 The ligated vectors were observed in well 4, 5 and 6

### Week 7

* Visualizing MAM7 ligated pET-22b(+)
* Transformation of MAM7 ligated pET-22b(+) to *E. coli* DH5α by PEG method (Trial 2)

**Troubleshoot**
 Since the competent
cells we prepared were a day old despite being stored at -80°C. Hence we
assumed
this to be the reason for our failed transformation

* Competent Cell preparation using the CaCl2 method (Trial 1)
* Transformation of MAM7 ligated pET-22b(+) to *E. coli* DH5α by CaCl2 method
  (Trial 1)
* Competent cell preparation (Trial 2)
* Colony PCR (Trial 1 of CaCl2 method) = 5 mixtures from 1:3

**Result**
 -The vector to insert ratios
yielded proper

 - Colonies 1,2,4,6 and 10 showed the best

 - The MAM7 amplicon band (At around 2.5kb) was distinct for these colonies and showed the
least number of non-specific bands.

* Running of gel for Colony PCR (Trial 1)

**Result**
 No insert was observed.
Instead, a big smear was observed at that location.

* Transformed pET-22b(+) plasmid Isolation using Miniprep Kit

**Result**
 During the gel running, two
bands were observed corresponding to one circular and one linear
plasmid. But on checking for the concentration, the value was too low.

* Colony PCR (Trial 2 of CaCl2 method) = 3 mixtures from 1:2, 4 reaction mixtures
  from
  1:3
  transformed plates.
   -> 3 reaction mixtures from 1:2 transformed plates

   -> 4 reaction mixtures from 1:3 transformed plates

**Result**
 After running the gel, we
observed 3 bands, out of which we observed one band exactly below the
insert. The reason for the other two nonspecific bands observed is yet to be troubleshot.

* Transformed pET-22b(+) Isolation using Miniprep Kit

**Result**
 We obtained a good
concentration of the plasmid. After running the gel, we could observe only one
single linear band.

* Single digestion of the transformed pET-22b(+) ligated with MAM7 using NdeI was carried out.
* We ran the gel and confirmed that our enzymes were working.
* Double Digested transformed pET-22b(+) ligated with MAM7 using NdeI and XhoI Enzymes
* Gel extraction was performed

**Result**
 - 3/4th of the mixture was
subjected to gel electrophoresis.
 - After gel extraction, we obtained the concentration of
pET-22b(+) and MAM7 after double
digestion i.e. 5.5ng/μL and 5.6ng/μL, respectively.

* PCR Purification

**Result**
 - We obtained higher
concentrations via PCR purification than Gel extraction.
 - pET-22b(+) =17.1ng/μL,
17.7ng/μL=MAM7; were the obtained concentrations.

* pET-22b(+) and MAM7 Ligation

   - Higher ratios for vectors to insert were considered

   - Vector to Insert ratios that were ligated = 1:4(Gel Extracted), 1:5, 1:6(PCR Purified)
* Competent Cell preparation using the CaCl2 method (Trial 2)
* Transformation of MAM7 ligated pET-22b(+) to *E. coli* DH5α by CaCl2 method
  (Trial 2)

![Single Digestion of transformed pET22-b+](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-single-digest.jpg)

Single digestion of transformed pET-22b(+)

![Double Digestion of transformed pET22-b+](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-double-digest.jpg)

Double digestion of transformed pET-22b(+)

![2nd Ligation of MAM7 insert and pET22-b+ vector](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-ligation-try-2.jpg)

2nd Ligation of MAM7 insert and pET-22b(+) vector

 The ligated vectors were observed in well 5

![pET-22b+ plasmid isolation.](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-isolated-plasmid.jpg)

pET-22b(+) plasmid isolation.
 Vector bands were
observed in well number 2 and 3

![Colony PCR Trial 1](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-colony-pcr-try-2.jpg)

Colony PCR Trial 1

![Colony PCR Trial 2](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-colony-pcr-try-1.jpg)

Colony PCR Trial 2

![Transformed plates using CaCl2- Trial 1](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-cacl2-trial-2.png)

Transformed plates using CaCl2- Trial
1

![Transformed plates using CaCl2- Trial 2](https://static.igem.wiki/teams/4200/wiki/wet-lab-timeline/week-7-cacl2-trial-1.png)

Transformed plates using CaCl2- Trial
2

# Biosafety

Given the conversations we had with the experts in domains like Biosafety and Biosecurity,
we decided to not work
with the risk-2 organism *Vibrio parahaemolyticus*. Instead, we decided on expressing our target protein– MAM7
in
our chassis– *E. coli* and performing further experiments with it. Here we are making use of the truncated
version
of MAM7, lacking the first 44 amino acids which are required for its membrane localization in its pathogenic origin.
Thereby the modified MAM7, when heterologously expressed in *E. coli*, will not be able to mediate any
harmful effects. This allowed us to avoid the risks of disease transmission to human beings and environmental
dissemination, while complying with the laws and regulations of recombinant research provided by the university.

Further, in order to gain some on-sight experience, we visited Dr. Mamatha Ballal’s lab
i.e., the Enteric Diseases
Division. Here we were provided with the opportunity to witness the culturing and maintenance of *V.
parahaemolyticus* stocks in a supervised environment.

**References**

[1] Benchling [Biology Software]. (2022). Retrieved from https://benchling.com.

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