iGEM 2023 — Team nyu-abu-dhabi

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{% block title %}Notebook{% endblock %}
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# Notebook

# July

# July 23

Prepared LB Agar Plate and Broth containing appropriate antibiotics for the Interlab study.

# July 24

##### Started InterLab Study 1 and 3 Day 0 protocol

###### Transformation protocol used:

1. Obtain agar plates and label them appropriately.
2. Aliquot 25 uL of competent E. coli DH5-alpha into an Eppendorf tube (Initial volume in each tube contains
   50).
3. Add 2 uL of plasmid into the first tube containing the competent E. coli. Allow the stored resuspended
   distribution kit to thaw before use. Store kit back at -20°C.
4. Place mixture on ice for 20 minutes.
5. Heat-shock the bacteria for 60 seconds using a heat block at 42°C.
6. Immediately place tubes on ice for 2 minutes.
7. Add 970 uL of SOC.
8. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
9. Spread gently using a plastic spreader.
10. Incubate overnight (16 hours) at 37°C.

# July 25

Transformations were unsuccessful. Possibly because the final solution plated had too
much SOC and, upon filling the plate, the solution flowed off the plate.

---

### July 26 – August 29: Summer break

---

# August

# August 30

1. Weighed out 8.75 g of LB Agar and 10 g of LB Broth and added it to 250 mL and 500 mL of diH2O respectively.
2. Sealed the flask with aluminum foil and put autoclave tape on top.
3. Placed both flasks in the autoclave. Ensured the water level inside the autoclave was sufficient and the
   water level was below the low marking on the water tank at the bottom right.
4. Started the autoclave using Mode 4. Flasks can be left in the autoclave overnight because of the keep warm
   feature (50°C).

# August 31

###### Preparation of Agar Plates and Broth

1. 250 uL of antibiotic was added to the pre-prepared LB Agar.
2. Added appropriate antibiotic to the LB Agar solution.
3. Prepared and filled 20 Petri dishes with the LB Agar.
4. Petri dishes were left to dry.
5. LB Broth that was left overnight was foamy, and signs of contamination were detected, so a new broth was
   made.

###### Transformation by Heat Shock

1. Obtain agar plates and label them appropriately.
2. Aliquot 25 uL of competent E. coli DH5-alpha into an Eppendorf tube (Initial volume in each tube contains
   50).
3. Add 2 uL of plasmid into the first tube containing the competent E. coli. Allow the stored resuspended
   distribution kit to thaw before use. Store the kit back at -20°C.
4. Place mixture on ice for 20 minutes.
5. Heat-shock the bacteria for 60 seconds using a heat block at 42°C.
6. Immediately place tubes on ice for 2 minutes.
7. Add 970 uL of SOC.
8. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
9. Centrifuge for 2 minutes at 13,000 rpm and discard 900 uL of supernatant to concentrate the bacteria.
10. Carefully resuspend the pellet by pipetting up and down and transferring 50uL of transformed bacteria to the
    respective agar plate containing the appropriate antibiotic.
11. Spread gently using a plastic spreader.
12. Incubate overnight (16 hours) at 37°C.

---

# September

# September 1

1. Checked on the transformation to verify successful transformation. 3 plates did not have
   any growth on them.
2. LB Broth was contaminated again. The broth (500 mL) was prepared again using a new
   LB Broth powder.

# September 4

1. Received training for Biotek Cytation 5 microplate reader and Excella Shaker with Sneha
   Ann Thomas, CTP Molecular and Cell Biology
2. Performed calibration portion of the InterLab Study.

# September 5

1. Repeated transformations for Interlab study for wells 13A, 13C, 13G.
2. Inoculated the transformations that worked into liquid broth following the InterLab
   protocol.

# September 6

1. Transformations still didn’t work for Interlab. Growth was observed on the positive
   controls. Constructs in the well could have been degraded because of storage over the
   break.

# September 7

1. Prepared agar plates with kanamycin

###### Resuspension of lyophilized plasmid

1. Keep the vial sealed and on ice until ready to use.
2. Centrifuge at 6,000x g for 1 minute at 4°C.
3. Resuspend pQE-80L-Kan in 10 uL of IDTE.
4. Resuspend pUC57-Kan containing collagen-like region from Scl2(CO) in 20 uL of nuclease-free water.
5. Keep both tubes on ice and proceed with the transformation of competent DH5 alpha cells.

###### Streak E. coli Auxotroph strain

E. coli Keio Knockout Strain (glycerol stock) JW id: JW0233 Strain: 2 ECK number:
ECK0244 Escherichia coli MG1655 B id: b0243 Gene name: proA

E. coli Keio Knockout Strain (glycerol stock) JW id: JW2806 Strain: 1 ECK number:
ECK2836 Escherichia coli MG1655 B id: b2838 Gene name: lysA

* Streak bacteria following [Addgene’s protocol.](https://www.addgene.org/protocols/streak-%20%20%20%20%20%20%20%20%20%20plate/)

# September 8

1. Transformation from yesterday did not work.
2. Repeated transformations for pQE-80L-Kan and pUC57-Kan containing the collagen-like
   region from Scl2(CO). Positive control was included to check if the error was in the
   protocol.

# September 10

1. Both transformations worked.
2. Multiple individual colonies were picked and inoculated into individual tubes containing 6
   mL of LB Broth with Kan.

# September 11

1. Performed MiniPrep for pQE80L-Kan and pUC57-Scl2.28 following the standard protocol
   listed on the experiments page.
2. Performed Nanodrop measurements for the samples. The yield of the plasmid in (ng/uL)
   was noted on the tubes. 260/280 were in the appropriate range.

# September 12

1. Prepared 1% agarose gel.
2. Double digest of pQE80L-Kan and pUC58-Scl2.28 using BamHI-HF and HindIII-HF for
   25 minutes.
3. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest, pUC57-
   Kan with Scl2.28, pUC57-Kan with Scl2.28 double digest using appropriate ladder.
4. Successful double digestion of pQE80L-Kan could not be determined. More details on
   [engineering success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27wiki_index%27%29%20%7D%7D) page.

# September 13

1. Prepared 1% agarose gel.
2. Double digest of pQE80L-Kan was repeated following the same procedure but for a
   different tube of Miniprepped plasmid.
3. pUC57-Scl2.28 using single digested HindIII-HF to determine size of linearized plasmid.
4. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest,
   pQE80L-Kan Sample 2, pQE80L-Kan Sample 2 double digest, pUC57-Kan with Scl2.28
   double digest, and pUC57-Kan with Scl2.28 single digest.

# September 14

1. Prepared 3% agarose gel.
2. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest, and
   pQE80L-Kan double digest S2 to check for fragments between the restriction sites.
3. Double digestion of pQE80L with BamHI and HindIII for 1 hour and 2 hours. Digestion
   was also performed sequentially BamHI followed by HindIII for 30 minutes each.
4. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest 1hr,
   pQE80L-Kan double digest 2hr, and pQE80L-Kan single digest.

# September 15

1. Single colonies of pQE80L in DH5-alpha were incubated in 5 mL of LB Broth.

# September 21

1. Plasmid miniprep of overnight culture of pQE80L.
2. Nanodrop of extracted plasmid.
3. Two different samples of double digested pQE80L were ligated with Scl2.28 using T7
   ligase.

# September 22

1. E. coli BL21 was transformed using ligated product.

# September 23

1. Plasmid was extracted from overnight liquid culture through Miniprep.
2. Extracted plasmid purity was measured using Nanodrop.
3. Extracted plasmid was double digested using BamHI and HindIII to verify the correct
   sequence.
4. Gel electrophoresis of extracted plasmid and double digested product.

# September 24

1. pQE80L double digested for 30 min, 1 hour, and 2 hour was ligated to double digested
   pUC57-Kan with Scl2.28 using T7 ligase.
2. Each plasmid was used to transform BL21.

# September 25

1. Miniprep of pQE80L-Kan overnight liquid culture.
2. The purity of extracted plasmid was quantified using Nanodrop.

# September 26

1. Single colonies were observed on all three combinations.
2. pQE80L digested for 20 mins using BamHI followed by 20 mins using HindIII.
3. Ligation of the two successive single digested pQE80L and double digested pQE80L with
   Scl2 was performed using T4 Ligase.
4. Transformation into DH5-alpha.

# September 27

1. Prepared 500 mL of LB Broth with Kan.
2. Inoculated single colonies of each construct into LB Broth..

# September 28

1. Miniprep of liquid culture was performed using standard procedure.
2. Nanodrop was used to quantify the purity of plasmid DNA.
3. PCR of plasmid to verify successful ligation.
4. Gel electrophoresis of plasmid and PCR product.

# September 30

1. PCR amplification of fragments 1-4 of Scl2.28 for Gibson Assembly.
2. PCR amplification of ProS and LysA gene from the two auxotroph strains.
3. Inoculated.

---

# October

# October 1

1. Miniprep of liquid culture was performed using standard procedure.
2. Nanodrop was used to quantify the purity of plasmid DNA.

# October 5

1. Fragment 4 was amplified for Gibson Assembly using higher annealing temperature.

# October 6

1. Gibson assembly of PCR amplified fragments.

# October 7

1. Transformation of BL21 with pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and
   pQE80L-Kan + Scl2.28 T4 Ligated.

# October 8

1. Preparation of LB Agar and LB Broth containing Kanamycin.
2. Single colonies of transformed BL21 were inoculated in LB Broth containing Kanamycin.
3. Preparation of competent E. coli auxotroph strain JW2806 and JW0233 using calcium chloride solution.
4. Transformation of auxotroph strain using pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan
   + Scl2.28 T4 Ligated.

# October 9

1. Miniprep of overnight liquid broth of BL21 transformed by pQE80L-Kan 2hr Double Digested + Scl2.28 T7
   Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.
2. 4 mL of overnight starter culture of BL21 was added to 100 mL of LB Broth containing Kanamycin.
3. IPTG was added to a final concentration of 1 mM once the OD reached 4-5.
4. Growth was observed on positive control for JW2806 and JW0233 and Cam plates for both plasmids. Single
   colonies were not distinguishable in Kanamycin plates.
5. Half of the induced cultures were pelleted at 6 hours.

# October 10

1. Remaining half of the induced cultures were pelleted after 18 hours.
2. Preparation of 1.5% agarose gel.
3. Preparation of LB broth.
4. Preparation of buffer for protein purification.
5. Double digestion of pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.
6. PCR to verify successful ligation of pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan +
   Scl2.28 T4 Ligated.

# October 11

* Purification of Scl2.28 from cell pellet using Qiagen Ni-NTA Spin Kit.
* SDS-PAGE of fraction from protein purification.

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4812/wiki/nyuad-igem-2023-lab-notebook.pdf%20%20%20%20%20%20%20%20%20%20)
instead.

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{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
{% block page\_content %}

# Engineering Success

---

# In a Nutshell

#### In the process of planning, developing, and executing our iGEM project to produce biomimetic collagen-like protein, the engineering design cycle was instrumental. This cycle consists of several iterative steps that help in defining problems, generating solutions, and implementing those solutions effectively. Here's how the engineering design cycle was applied to our project.

# Gibson Assembly

## Design

The collagen-like region from Scl2.28 (AY069936) we chose as our protein
of interest had repeated sequences, which lead to a high complexity score.
As such, we were unable to place the order for synthesis from IDT. Hence,
we considered using Gibson Assembly for assembling the plasmid. For this
we fragmented the original sequence into four different parts. The E. coli
proline (JW0233) and lysine (JW2806) auxotroph strains are derived from
K12, so we codon-optimized the fragments. By doing so, we were able to
synthesize the gene of interest.

## Original Scl2

```
      CAAGACGGTCGAAACGGTGAAAGAGGGGAACAAGGACCAACAGGTCCAACCGGACCTGCTGGTCCACGAGGTCTACAAGGTCTACAAGGTCTACAAGGTGAAAGAGGGGAACAAGGACCAACAGGTCCCGCTGGTCCACGAGGTCTACAAGGTGAAAGAGGGGAACAAGGACCAACAGGTCTCGCT
```

```
   GGTAAAGCCGGTGAAGCTGGAGCCAAAGGCGAAACCGGCCCCGCTGGTCCACAGGGTCCACGTGGTGAACAAGGCCCGCAAGGTCTTCCAGGTAAAGATGGTGAAGCTGGTGCTCAAGGCCCAGCAGGTCCAATGGGTCCTGCTGGTGAGCGAGGTGAAAAAGGAGAACCTGGTACCCAAGGCGCTAAAGGT
```

```
    GATCGCGGTGAAACCGGTCCAGTAGGTCCACGTGGTGAGCGAGGCGAAGCCGGTCCCGCTGGAAAAGATGGTGAACGTGGTCCAGTAGGTCCAGCTGGTAAGGACGGCCAAAACGGCCAAGATGGTCTTCCAGGTAAAGACGGTAAGGACGGCCAAAACGGTAAAGATGGTCTTCCAGGTAAAGACGGTAAGGAC
```

```
    GGCCAAAACGGTAAAGATGGTCTTCCAGGTAAAGACGGTAAGGACGGTCAAGATGGTAAAGACGGCCTCCCAGGTAAAGACGGTAAAGATGGCCTCCCAGGTAAGGACGGTAAGGACGGTCAACCAGGTAAACCGGCT
```

#### Optimized for E. coli K12

```
    CAAGACGGTCGTAATGGCGAACGCGGCGAACAGGGGCCTACAGGCCCCACTGGCCCAGCCGGGCCACGCGGTCTTCAGGGACTGCAAGGGTTGCAAGGTGAGCGTGGAGAACAGGGTCCTACCGGGCCAGCAGGTCCCCGTGGACTTCAGGGTGAACGTGGGGAGCAAGGTCCCACCGGCTTAGCT (Total Complexity Score: 8.1)
```

```
    GGTAAAGCGGGGGAAGCTGGTGCAAAAGGAGAGACGGGTCCAGCCGGTCCCCAGGGCCCACGTGGTGAGCAAGGCCCCCAAGGCTTACCTGGCAAGGATGGCGAGGCTGGCGCGCAAGGCCCGGCAGGCCCAATGGGACCCGCAGGAGAACGCGGAGAAAAGGGTGAACCAGGCACTCAAGGTGCCAAAGGT (Total Complexity Score: 7.6)
```

```
    GATCGCGGAGAAACGGGACCGGTCGGTCCTCGTGGAGAACGTGGGGAAGCAGGCCCCGCAGGGAAGGATGGTGAGCGTGGCCCCGTTGGACCAGCCGGAAAAGATGGGCAGAACGGCCAGGACGGCTTACCGGGGAAAGACGGAAAAGATGGCCAAAATGGTAAGGATGGTTTACCAGGGAAAGATGGTAAGGAC (Total Complexity Score: 7.4)
```

```
    GGCCAGAATGGGAAGGACGGGTTGCCAGGAAAAGACGGCAAAGACGGACAAGATGGTAAAGATGGACTGCCGGGCAAGGATGGAAAGGATGGCTTACCGGGAAAAGACGGTAAGGATGGACAGCCGGGCAAGCCAGCT (Total Complexity Score: 7.6)
```

We used NEB’s Assembly tool to generate the primers needed for Gibson
Assembly for each fragment. The file from the tool can be accessed here.
The minimum overlap was specific at 25 nt and minimum primer length as 18
nt.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/gibson-assembly.png)

# Gibson Assembly

## Build

We used PCR to amplify the fragments and generate the overhangs needed for
Gibson Assembly with the denaturation at 95°C, annealing at 62°C, and
extension at 72°C.

## Test

To verify whether the fragments were successfully generated, we performed
gel electrophoresis with a 0.75% agarose gel. From the gel, we were able
to identify the bands for the 3 fragments, but not for the fourth
fragment. The 500 bp ladder was inappropriate for this gel electrophoresis
run.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/pcr-gibson-1.png)

## Learn

To identify the error, we went back to the NEB Assembly tool and realized
that the annealing temperature of the primers for the fourth fragment
(particularly the reverse primer) was much higher than the three other
fragments as shown in the picture below.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/oligos.png)

We redid PCR for the fourth fragment at an increased annealing an
extension temperature of 70°C and 75°C, keeping other parameters the same.
We reran the gel with an appropriate ladder and the fragment was visible
(Lane 5). Lane 6 shows the PCR product from the previous run.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/pcr-gibson-2.png )

---

# Restriction Digestion

## Design

In terms of experimental design, we choose to perform cloning via
restriction enzyme double digestion. Our gene of interest (Scl2.28) was
synthesized by GenScript and inserted into pUC57-Kan with BamHI and
HindIII cut site flanking the insert. BamHI and HindIII cut sites were
present in the MCS of the vector (pQE80L-Kan)

## Build

We performed double digestion of our vector (pQE80L-Kan) using BamHI-HF
(NEB, R3136) and HindIII-HF (NEB, R3104). As per NEB’s restriction enzyme
single/double digestion tool, we performed the double digestion at 37°C
for 15 minutes.

## Test

To verify successful double digestion before proceeding with ligation, we
performed gel electrophoresis with a 1% agarose gel. From the gel, we were
able to clearly identify the two fragments for the pUC57-Kan indicating
successful digestion. The insert should be 717 bp long, and we saw
corresponding band. For the vector (pQE80L-Kan) it was difficult to
identify successful digestion because the region between the two
restriction enzymes was only 42 bp long.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/double-digest-1.png )

## Learn

Since we were unsure of double digestion, we decided to redo the double
digestion for the vector. The plasmid with the insert travelled further
than expected most likely because of being supercoiled. Hence, we decided
to perform a single digest to identify the actual size of the plasmid. The
single digested pUC57-Kan (3260 bp) showed up in the expected range in the
gel. For the vector, we still weren’t able to see any significant
difference between the undigested and digested plasmid. The fragment in
between the restriction sites were also not visible while running the gel
with a 100 bp ladder and higher percentage of agarose. We hypothesized
that the timing for the digestion was not enough and decided to perform
double digestion for longer.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/gel-2.png )
![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/gel-3.png)

## Design/Build

Learning from the previous iteration, we performed double digestion of the
vector for 1 hour and 2 hours. Single digestion was also performed to
characterize the size of the vector.

## Test

The gel electrophoresis of double digested plasmid for both the timings
were similar to previous iteration and was not conclusive. The single
digested plasmid (4541 bp) was in similar range to the undigested plasmid.

![Image Alt Text](https://static.igem.wiki/teams/4812/wiki/engineering-success/gel-4.png)

## Learn

We decided to consult Dr. N. Ayyadurai (Principal Scientist at
CSIR-Central Leather Research Institute, Chennai, India), whose
influential paper on incorporation of hydroxyproline into bacterial
collagen like protein, we were referencing. During the consultation, he
mentioned that the moustache-like overhangs we were observing in the
double digested plasmid was indicative of successful digestion. With this
knowledge, we decided to proceed with ligation and transformation.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

# Quick Links

### [Cloning Workflow](#cloning)

### [Purification of CLTP using Qiagen Ni-NTA Fast Start Kit](#faststart)

### [Purification of CLTP](#purification)

### [General Molecular Biology Protocol](#general)

---

# Cloning Workflow

##### Resuspension of lyophilized plasmid

##### +

1. Keep the vial sealed and on ice until ready to use.
2. Centrifuge at 6,000x g for 1 minute at 4°C.
3. Resuspend pQE-80L-Kan.in 10 uL of IDTE.
4. Resuspend pUC57-Kan containing collagen-like region from Scl2(CO) in 20 uL of nuclease-free water.
5. Keep both tubes on ice and proceed with transformation of competent DH5 alpha cells.

##### Transformation by Heat Shock

##### +

1. Obtain agar plates with KAN antibiotic and label them appropriately.
2. Allow competent E. coli DH5-alpha or BL21 to thaw on ice. Aliquot 25 uL of DH5-alpha into an Eppendorf tube
   (Initial volume in each tube contains 50). For BL21, use the entire tube.
3. Add 1-5 uL of plasmid into the first tube containing the competent E. coli.
4. Place mixture on ice for 20 minutes.
5. For DH5-alpha, heat-shock the bacteria for 60 seconds using a heat block at 42°C. For BL21, heat-shock the
   bacteria for exactly 10 seconds using a heat block at 42°C.
6. Immediately place tubes to ice for 2 minutes for DH5-alpha and 5 minutes for BL21.
7. Add 970 uL of SOC.
8. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
9. Centrifuge for 2 minutes at 4000 rpm and discard 900 uL of supernatant to concentrate the bacteria.
10. Carefully resuspended the pellet by pipetting up and down and transferring 50uL of transformed bacteria to
    the respective agar plate containing the appropriate antibiotic.
11. Spread gently using plastic spreader.
12. Incubated overnight (16 hours) at 37 °C.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/protocols/transformation.png )

##### Growth of bacterial cultures in tubes or flasks

##### +

1. Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium
   containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking.
   * Growth for more than 16 h is not recommended because cells begin to lyse and plasmid DNA yields may be
     reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture.- Harvest the bacterial cells by centrifugation at >8000 rpm (6800 x g) in a conventional table-top
     microcentrifuge for 3 min at room temperature.
     * The bacterial cells can also be harvested in 15 ml centrifuge tubes at 5400 x g for 10 min at 4°C.
       Remove all
       traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

###### Buffer notes

##### +

1. Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A (centrifuge briefly before
   use) per bottle Buffer P1 for a final concentration of 100 µg/ml. Mix and store at 2–8°C.
2. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
3. Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to
   37°C. Do
   not shake Buffer P2 vigorously.
4. Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from CO2
   in
   the air.
5. Buffers P2, N3 and PB contain irritants. Wear gloves when handling these buffers.
6. **Optional:** Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue
   reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 µl LyseBlue into 10 ml Buffer P1).
   LyseBlue provides visual identification of optimum buffer mixing, thereby preventing the common handling
   errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell
   debris.

##### Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and a Microcentrifuge

##### +

This protocol is designed for the purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight
cultures of Escherichia coli in LB medium. **Note: All protocols steps at room temperature.**

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
   * Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of
     the pellet.
   * If LyseBlue reagent has been added to Buffer P1, before use vigorously shake the buffer bottle to ensure
     LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or
     pipetting up and down until no cell clumps remain.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
   * Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and
     contamination of plasmid. If continue inverting the tube until the solution becomes viscous and slightly
     clear. Do not allow the lysis reaction to proceed for more than 5 min.
   * If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after the addition of Buffer
     P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized
     colorless regions, or if brownish cell clumps are still visible, continue mixing the solution until a
     homogeneously colored suspension is achieved.
3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times.
   * To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3.
     Large culture volumes (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become
     cloudy.
   * If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue is gone and the
     suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively
     precipitated.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet
   will form.
5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
6. Centrifuge for 30–60 s. Discard the flow through.
7. **Recommended:** Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60
   s. Discard the flow through.
   * This step is necessary to remove trace nuclease activity when using endA+ strains, such as the JM series,
     HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high
     carbohydrate content. Host strains, such as XL-1 Blue and DH5α, do not require this additional wash step.
8. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
   * **Important:** Residual wash buffer will not be completely removed unless the flow-through is
     discarded
     before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic
     reactions.
10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB
    (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and
    centrifuge for 1 min.
11. Measure DNA concentration using NanoDrop.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/protocols/miniprep.png)

##### Restriction Enzyme Double Digestion

##### +

1. Set up the reaction as follows:

   | Component | 50 µl REACTION |
   | --- | --- |
   | DNA | 1 µg |
   | 10X rCutSmart Buffer | 5 µl (1X) |
   | BamHI-HF | 1.0 µl (20 units) |
   | HindIII-HF | 1.0 µl (20 units) |
   | Nuclease-free Water | to 50 µl |
2. Incubate at 37°C for 5-15 minutes as both enzymes are Time-Saver qualified.
3. Heat inactivate at 80°C for 20 minutes.

##### Ligation

##### +

##### T7 DNA Ligase (M0318, NEB)

##### +

1. Set up the following reaction in a microcentrifuge tube on ice.

   (T7 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3
   vector
   to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.

   | Component | 20 µl REACTION |
   | --- | --- |
   | T7 DNA Ligase Buffer (2X)\* | 10 μl |
   | Vector DNA (4 kb) | 50 ng (0.020 pmol) |
   | Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |
   | Nuclease-free water | to 20 μl |
   | T7 DNA Ligase | 1 μl |

   \* The T7 DNA Ligase Buffer should be thawed and resuspended at room temperature.
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. Incubate at room temperature (25°C) for 15-30 minutes.
4. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Alternatively, store at
   –20°C.

##### T4 DNA Ligase (M0318, NEB)

##### +

1. Set up the following reaction in a microcentrifuge tube on ice.

   (T7 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3
   vector
   to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.

   | Component | 20 µl REACTION |
   | --- | --- |
   | T4 DNA Ligase Buffer (10X)\* | 2 μl |
   | Vector DNA (4 kb) | 50 ng (0.020 pmol) |
   | Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |
   | Nuclease-free water | to 20 μl |
   | T4 DNA Ligase | 1 μl |

   \* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours
   (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
5. Heat inactivate at 65°C for 10 minutes.
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

##### HiFi DNA Assembly Protocol

##### +

1. Set up the following reaction on ice.

   |  | 2–3 Fragment Assembly\* | 4–6 Fragment Assembly\*\* | NEBuilder Positive Control✝ |
   | --- | --- | --- | --- |
   | Recommended DNA Molar Ratio | vector:insert = 1:2 | vector:insert = 1:1 |  |
   | Total Amount of Fragments | 0.03–0.2 pmols\* X μl | 0.2–0.5 pmols\*\* X μl | 10 μl |
   | NEBuilder HiFi DNA Assembly Master Mix | 10 μl | 10 μl | 10 μl |
   | Deionized H2O | 10-X μl | 10-X μl | 0 |
   | Deionized H2O | 20 μl✝✝ | 20 μl✝✝ | 20 μl |

   \*Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar
   excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction
   should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each
   fragment.

   \*\*To achieve optimal assembly efficiency, design 20-30 bp overlap regions between each fragment with
   equimolarity of all fragments (suggested: 0.05 pmol each).

   ✝Control reagents are provided for 5 experiments.

   ✝✝If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional
   NEBuilder HiFi DNA Assembly Master Mix.
2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60
   minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for
   subsequent transformation.
3. Transform NEB 5-alpha or 10-beta Competent E. coli cells with 2 μl of the chilled assembled product, following
   the transformation protocol.

---

# Purification of CLTP using Qiagen Ni-NTA Fast Start Kit

##### ([Source](https://www.qiagen.com/us/products/discovery-and-translational-research/protein-purification/tagged-protein-expression-purification-detection/ni-nta-fast-start-kit%20))

##### Growing E. coli Cells and Inducing

##### +

1. Using a sterile toothpick or inoculating loop, pick a single colony from a selective antibiotic LB agar
   plate and inoculate 10 ml of LB medium containing 50 μg/ml kanamycin in a 50 ml flask. Grow the cultures at
   37°C overnight.
2. Inoculate 150 ml of prewarmed media (with ampicillin) with 4 ml of the overnight cultures and grow at 37°C
   with vigorous shaking until an OD600 of 0.6 is reached (30–60 min).
3. Take a 0.5 ml sample immediately before induction for noninduced control; pellet cells and resuspend them in
   25 μl 5x SDS-PAGE sample buffer. Freeze and store the sample at –20°C until SDS-PAGE analysis.
4. Induce expression by adding IPTG to a final concentration of 1 mM.
5. Incubate the cultures for an additional 6 hour and overnight (16 hour). Collect a second 0.5 ml sample as
   the induced control; pellet cells and resuspend them in 50 μl 5x SDS-PAGE sample buffer. Freeze and store the
   sample at –20°C until SDS-PAGE analysis.
6. Harvest the cells by centrifugation at 4000 x g for 20 min.
7. Freeze and store cell pellet overnight at –20°C.

##### Purification under denaturing condition

##### +

1. Resuspend the cell pellet in 10 ml Denaturing Lysis Buffer.
2. Incubate at room temperature (15–25°C) for 60 min. Mix 2–3 times by gently swirling the cell suspension.
   Lysis is complete when the suspension is translucent.
3. Centrifuge lysate at 14,000 x g for 30 min at room temperature (15–25°C) to pellet the cellular debris.
4. Retain the cell lysate supernatant. The supernatant contains the recombinant protein.
5. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of the supernatant and store at –20°C for SDS-PAGE
   analysis.
6. Gently resuspend the resin in a Fast Start Column by inverting it several times.
7. Break the seal at the outlet of the column, open the screw cap, and allow the storage buffer to drain out.
   It is important that the outlet seal is broken before the screw cap is removed.
8. Apply the cell lysate supernatant from step 3 to the column.
9. Collect the flow-through fraction. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of the flow-through
   fraction and store at –20°C for SDS-PAGE analysis.
10. Wash the column 2 times with 4 ml of Denaturing Wash Buffer.
11. Collect both wash fractions. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of each wash fraction and
    store at –20°C for SDS-PAGE analysis.
12. Elute bound 6xHis-tagged protein with two 1 ml aliquots of Denaturing Elution Buffer.
13. Collect each elution fraction in a separate tube. Add 5 μl 2x SDSPAGE sample buffer to a 5 μl aliquot of
    each elution fraction and store at –20°C for SDS-PAGE analysis.
14. Analyze all fractions by SDS-PAGE.

---

# Purification of CLTP

### Modified by Rashik Chand for NYUAD iGEM,

##### Original Source: Samar Fadi and Juliana Ferreira, Rabeh Lab, NYUAD

### Day 1

##### Steps

##### +

1. Start overnight seed culture at 37 °C:
2. 50ml LB containing antibiotics (pQE80L-KAN-Scl2.28) KAN and CHL.
3. Samples of seed culture in 50ml LB containing:
   * Kanamycin-KAN (50 ug/mL)
   * Chloramphenicol-CHL (25 ug/mL)

### Day 2

##### Inoculation

##### +

1. Clean the bench and pipettes with alcohol, turn on the fire.
2. Creating 4L of each sample : Grab 1 L of L-Broth in 4 flasks each.
3. Add 500 uL of KAN and CHL each to every flask (1:500 antibiotics:LB ratio). Kan (50 mg/L) and CHL(25 mg/L).
4. Add 10 ml of pre-inoculated bacterial culture to each flask
5. Transfer everything to the shaking incubator (Temperature:37℃ , RPM: 200) for 4 hours. Check if they are
   stable and properly stuck to the pads.
6. **Checking optical density using spectrophotometer:**
   * Grab 2 cuvettes (one for blank and one for the sample)
   * Clean the bench and pipettes with alcohol, turn on the fire.
   * Add (less than 5 ml- filling 3/4th cuvette ) blank: plain L-Broth used for inoculation and sample : from
     the flasks in shaking incubator using the pipette with glass tubes.
   * Switch on and run the Spectrophotometer software
   * Add the blank, on measuring the OD, press on auto-zero
   * Add the sample and measure the OD
   * If OD>=0.3, start inducing. Else continue incubating in the shaker.

##### Induction using IPTG

##### +

Isopropyl-ß-D-thiogalactopyranoside (IPTG) is a chemical analogue of galactose, which cannot be hydrolyzed by
the enzyme ß--Galactosidase. Hence, it induces the E. coli lac operon activity by binding and inhibiting the lac
repressor without being degraded.

1. Prepare an ice bath to store the IPTG
2. Grab IPTG from the -30 freezer
3. Clean the bench and pipettes with alcohol, turn on the fire.
4. Add 500uL of IPTG to the culture using autoclaved tips in aseptic conditions.
5. Transfer everything to the shaking incubator (Temperature: 37℃ , RPM : 120) for 18 hours.

##### Residue-specific incorporation of trans-4 hydroxyproline and hydroxylysine in CLTP

##### +

1. From the growth curve experiment, choose the optimum concentration of proline and lysine for
   residue-specific incorporation of Hyp and Hyl.
2. Inoculate single colony of proline/lysine auxotroph containing pQE-CLTP into Luria Bertani medium
   containing 100 μg ml−1 of kanamycin and incubate in a shaker at 37 °C overnight.
3. Inoculate 100 ml of adaptation medium (minimal medium with 40 μg ml−1 of proline) from the overnight grown
   culture, having 0.2 OD at 0th hour and incubated overnight at 37 °C. 1% of inoculum.
4. Prepare 1-liter minimal medium containing 0.04 mM of proline.
5. Inoculate overnight adaptation culture into the newly prepared 1 liter minimal medium, having 0.2 OD after
   inoculation.
6. After the culture reaches the stationary phase (OD: 0.9 - 1) 10–80 mM of trans-4 hydroxyproline (Hyp) was
   added along with 1 mM of IPTG.
7. Then 500 mM of NaCl was added to each medium and incubated for 7 h at 37 °C. The trans-4 hydroxyproline
   incorporation efficiency was analyzed in whole cell lysate using SDS-PAGE.
   * NaCl assists in intracellular accumulation and the incorporation of Hyp by assisting in transport of Hyp.
     It is unique to Hyp and may or may not be necessary for Hyl.

### Day 3

##### Steps

##### +

1. Cool the bacterial culture on ice before the centrifugation at 8000rpm for 10min at 4°C. Use the 1L
   centrifuge bottles. Make sure to balance your tubes to prevent centrifuge from damage and ensure safety.
2. Dissolve the cell pellet (1L overnight cell culture) in about 40 ml lysis buffer (0.5g of bacterial
   pellet/ml of lysis buffer).
   * Meanwhile, equilibrate the nickel column (3 ml resin/bed volume) in the cold room by 30ml lysis buffer.
3. Use homogenizer to dissolve the bacterial pellet in the lysis buffer. You can vortex to further dissolve the
   bacterial pellet.
4. Sonication in glass beaker. Sonication on for 5 sec, stop for 5 sec. Total sonication time is 5 min on time.
   30% power with large tip
5. Centrifuge the cell extract using at 18000 rpm for 45 min at 4 degrees.
6. Load the supernatant to 3 mL column bed volume of Ni-NTA by pumping 3 rpm on the peristaltic pump.
7. Wash the column by (1) 50 ml lysis buffer then (2) 30ml wash Buffer A, by pumping 5 rpm on the peristaltic
   pump. Collect both washes each in a single 50mL falcon tube.
8. Elute the His-tag protein by elution Buffer B, 1mL fractions each time, 10 fractions in total.
9. Check for protein in the elution fractions using Bradford, 100ul Bradford/3ml protein.

   Keep 10uL of each fraction to be used for SDS-PAGE analysis that will be done later. Pool fractions that have
   high blue color intensities. If your last fractions still have high blue color, you can collect more elution
   fractions to make sure all your protein target is out of the column.
10. Run the pooled fractions of CLTP from Ni-NTA column on 16/60 S200 Gel Filtration (GF) column overnight at 1
    mL/min. The GF column needs to be priorly equilibrated or a column wash step should be added prior to running
    the sample.
11. Next day, determine the protein concentration with Bradford assay then run SDS-PAGE analysis to evaluate the
    sample purity.

##### Buffers

##### +

##### Minimal Media

##### +

1. M9 salts
2. 20% glucose
3. 0.2 mM of \(MgSO\_4\)
4. 0.1 mM of \(CaCl\_2\)
5. 1 μg \(ml^{−1}\) of thiamine HCl
100 μg \(ml^{−1}\) of all amino acids except proline6. 100 μg \(ml^{−1}\) ampicillin (50 ug/mL in our case)
7. Various concentrations of proline (0.01 mM, 0.02 mM, 0.03 mM, **0.04 mM,** 0.05 mM) - 0.04 mM chosen
   from
   growth curve analysis

##### Binding Buffer

##### +

(250 ml/column or construct) you will also need 100mL of stock buffer to equilibrate your column prior to
loading your cell lysate.

1. 50 mM \(Na\_2HPO\_4·2H\_2O\)
2. 500 mM NaCl
3. 20 mM Imidazole to improve sample purity by preventing non-specific binding of other proteins.

Cool to 4˚C then adjust pH to 7.4

##### Lysis Buffer

##### +

(take 100 ml of stock buffer for each column/construct)

1. 10 mM Tris
2. 1 mM EDTA
3. 0.1% SDS
4. Lysozyme (0.2 mg/mL)

##### Washing Buffer

##### +

(take 50 ml of stock buffer for each column/construct)

1. 50 mM \(Na\_2HPO\_4·2H\_2O\)
2. 500 mM NaCl
3. 40 mM Imidazole

Cool to 4˚C then adjust pH to 7.4.

##### GF Buffer

##### +

1. 50mM \(PO\_4\) buffer pH 7.5 (CD analysis) or 20mM Hepes pH 7.5
2. 150 mM NaCl
3. 0.5 mM TCEP

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/protocols/protein-purification.png)

**SDS-PAGE: Continuous 12%** expected band (∼35 kDa)

---

# General Molecular Biology Protocol

##### Agarose Gel Electrophoresis

##### +

##### Prepare 1% Agarose Gel

##### +

1. Weigh 0.5 g of agarose
2. Measure 50 ml 1 x TAE buffer
3. Dissolve agarose in TAE buffer by heating in microwave for 1 minute or until agarose
   dissolves completely
4. Add 3μl of EZ –VISION IN- GEL for you (Binds DNA band and enables you to see DNA bands under UV light)
5. Prepare the gel case by placing the plastic comb into the gel cast
6. Pour your gel in the gel cast and let to cool

##### Prepare samples for loading

##### +

1. Add 1 μl Purple Loading Dye to 5μl of digested insert and vector.
2. Place gel inside the electrophoresis device and add 1 x TAE running buffer until gel is completely
   immersed.
3. Load 5 μl of 1 KB DNA Ladder in the first well.
4. Load each of your prepared samples on separate wells
5. Apply electrical current (135V) and let gel run for 20 minutes
6. From your gel from gel apparatus and take a picture of your gel under UV in the Gel Documentation
   Station

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/protocols/gel-electrophoresis.png)

##### SDS Page

##### +

1. For handcasting of 12% discontinuous polyacrylamide gels, add the following components to two 50 mL Falcon
   tubes.

   **Resolving gel:**

   1. 5.5ml of ddH2O
   2. 5ml of 1.5 M Tris with pH=8.8
   3. 0.2ml of 10% SDS
   4. 8.7ml of 30% Acryl/Bis

   Directly before casting the gel, add the following:

   1. 100μl of 10% APS
   2. 10μl TEMED

   **Stacking gel:**

   1. 6.1ml of ddH2O
   2. 2.5ml of 0.5 M Tris-HCl with pH=6.8
   3. 0.1ml of 10% SDS
   4. 8.7ml of 30% Acryl/Bis

   Directly before casting the gel, add the following:

   1. 50μl of APS
   2. 10μl of TEMED
2. As soon as the APS and TEMED, proceed as quickly as possible to the step of casting the gel. Be very careful
   for any delay will result in gel polymerization in the tubes and you will have to repeat the preparations.
3. Use 1ml pipettes to transfer the gel into the setting and 4 to 5 times should be enough for the resolving
   gel and 2 times for the stacking. Once you add the resolving gel, add a layer of water or isopropanol. Once,
   resolving gel is set, add the stacking gel and the comb.
4. Keep the excess in the falcon tubes. Do not discard. This will allow you to monitor the polymerization of
   the gel.
5. Add 5ul of sample to 5ul of 2X Laemmli sample buffer.
6. Load the sample onto the wells and run the SDS-PAGE for 40 minutes at 200 V.
7. Wash gel 3 times in distilled water for 5 minutes each.
8. Add EZBlue Gel staining reagent and agitate for one hour.
9. Rinse with water for 1 hour to overnight.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/protocols/sds.png )

##### Competent Cell using Calcium Chloride

##### +

##### [Source](https://www.mybiosource.com/learn/testing-procedures/cacl2-transformation-technique)

1. Prepare a small, overnight culture of the bacteria in LB broth. Grow at 37°C.
2. About 2 h before you are ready to begin the main procedure, use 1.0 mL of the overnight culture to inoculate
   100 mL of fresh LB broth. This culture is grown with rapid shaking at 37°C until it reaches roughly 5 x 107
   cells/ml. Corresponding to OD between 0.3 and 0.5. Do not allow the cells to reach stationary phase.
3. Take a 5 mL aliquot of each transformation reaction and transfer to sterile plastic centrifuge tubes. Cool
   on ice for 10 min.
4. Pellet the cells by spinning for 5 min at 5000g. It is necessary for the centrifugation to be performed at
   4°C.
5. Pour off the supernatant and resuspend cells in 25 mL of cold 0.1M CaCl2. Leave on ice for at least 20 min.
6. Centrifuge as in Step 3. You should observe a more diffuse pellet than previously. This is an indication of
   competent cells.
7. Resuspend the cells in 0.2 mL of cold 0.1M CaCl2. And aliquot 50 uL into 1 mL Eppendorf tubes.
8. To each tube add up to 0.1 mg of DNA, made up in a standard DNA storage buffer such as TE to a volume of 100
   mL. Leave on ice for 30 min.
9. Heat shock at 42°C using a water bath or heat block.
10. Return to ice fo 3 minutes.
11. Add 970 uL of SOC media.
12. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
13. Centrifuge for 2 minutes at 4000 rpm and discard 900 uL of supernatant to concentrate the bacteria.
14. Carefully resuspended the pellet by pipetting up and down and transferring 50uL of transformed bacteria to
    the respective agar plate containing the appropriate antibiotic.
15. Spread gently using plastic spreader.
16. Incubated overnight (16 hours) at 37 °C.

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# Results

# Quick Links

### [Choosing the appropriate vector](#choose)

### [Prepare vector-insert construct](#prepare)

### [Expression and Purification](#expression)

### [Transformation of E. coli auxotroph strain](#transformation)

### [Future Steps](#future)

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/results-discussion/flowchart.png )

Outline: Results and Discussion

# Choosing the appropriate vector

In order to synthesize the bacterial collagen-like protein (Scl2.28), we decided to use the pQE80L-Kan plasmid
from Qiagen. The plasmid places a 6XHis tag at the N-terminus of the protein of interest allowing subsequent
purification using Ni-NTA affinity chromatography. pQE80L was specifically chosen as it encodes a cis-lacIq gene,
which makes it not require the pREP4 repressor plasmid compared to other pQE vectors. While other vectors, such as
the pColdIII has been extensively used in literature [1, 2], the use of pQE80L is recommended for fibril formation
studies of bacterial collagen-like protein and the incorporation of ncAAs [3], in our case, hydroxyproline and
hydroxylysine.

Ideally, we would choose pQE80L with resistance to ampicillin, however, the only vector we could import on time
was pQE80L with Kanamycin resistance. This makes is slightly tricky as the E. coli Keio Knockout Collection [4]
from which we obtained the proline and lysine auxotroph strains, JW0233 and JW2806 replaces the deleted proA and
lysA gene with a kanamycin resistance cassette. FLP recognition target (FRT) recombination can be used to excise
the kanamycin resistance; however, this is not something that we could try.

# Prepare vector-insert construct

One of the challenges that we faced was the cloning of the gene of interest into the pQE80L vectors. Further
details are documented on the Engineering Success page. We considered both Gibson Assembly and cloning using
restriction enzymes. Part of the consideration was the practicality of the option as ordering of complete Scl2.28
was not possible through IDT due to high complexity. While we were able to amplify the fragments for Gibson
Assembly, we could not obtain an assembled plasmid with the pQE80L and the fragments in appropriate order using
NEB HiFI DNA Assembly. However, previous iGEM team (Collagene, 2021) has reported being able to assemble the
plasmid using Gibson Assembly. It may be the case that the assembly conditions needs to be optimized.

For cloning using restriction digestion, we had to go through several iterations of ligating the Scl2.28 with
various combinations of pQE80L-Kan. Specifically, we tried out the following combinations:

1. pQE80L double digested for 15 mins ligated to double digested pUC57-Kan with Scl2.28 using T7 ligase.
2. pQE80L double digested for 30 mins (A), 1 hour (B), and 2 hours (C) ligated to double digested pUC57-Kan with
   Scl2.28 using T7 ligase.
3. pQE80L digested for 20 mins using BamHI followed by 20 mins using HindIII ligated with double digested
   pUC57-Kan with Scl2.28 using T4 ligase.
4. pQE80L double digested for 1 hour ligated to double digested pUC57-Kan with Scl2.28 using T4 ligase.

\* BamHI-HF and HindIII-HF was used for all digestions.

Ligation reaction 1 and subsequent transformation in BL21 resulted in single colonies. To verify the construct,
plasmid amplification and purification was performed. The purified plasmid was double digested. The bands obtained
were the same as the bands for the pUC57-Kan with Scl2.28. This indicated to us that the digestion may not be
going to completion and residual pUC57-Kan could interfere with subsequent screening. Hence, the digestion time
was increased to 30 minutes to an hour for Scl2.28.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/results-discussion/ligation1.png
      )

Following this, we transformed the plasmids obtained in combinations 2A, 2B, 2C, 3, and 4. Subsequently, the
plasmids were amplified and double digested to check for desired band in gel electrophoresis as shown below. From
these gels, we shortlisted two possible candidates for further characterization, namely 2C and 4, as they had the
bands that matched double digested pQE80L-Kan (4500 bp) and the insert sequence of pUC57-Kan with Scl2.28 (711
bp). We theorized two possible scenarios for 4, one that the ligation was unsuccessful because of failed digestion
and both pQE80L-Kan and pUC57-Kan were present. The second scenario was that ligation was successful, but excess
pUC57-Kan was still present. The wide bands of the undigested ligated product also made it difficult to estimate
the size of the ligated plasmids. To get definitive verification of ligation, primers were designed and ordered
for PCR. The forward primer was on the insert sequence, while the reverse primer was on the vector backbone.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/results-discussion/ligation2.png
      )

For both the constructs (2C and 4), PCR product was visible. The PCR band was wide but fell in the appropriate
size (~800 bp). No PCR product was visible for negative control. This indicated successful generation of
pQE80L-Kan+Scl2.28 plasmid. These plasmids were then used to transform BL21 and used for subsequent expression.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/results-discussion/pcr-ligation.png
      )

# Expression and Purification

Overnight liquid culture of both 2C and 4 was added to 150 mL LB Broth and expression was induced using IPTG once
the OD600 reached 0.5. For each construct, one sample was allowed to incubate for 6 hours and one for 16 hours. At
the end of each duration, cells were pelleted. Cell pellets were used for purification following Qiagen Ni-NTA
Fast Start Kit protocols due to easy of preparing buffer and sample volume. The non-induced control, induced
control, cell lysate, flow through, wash fraction, and elution fractions were analyzed using SDS-PAGE followed by
Coomassie stain. A distinct band approximately 35 kDa corresponding to Scl2.28 is visible across all the SDS-PAGE
in the induced controls. This value is in close agreement with value from literature [3]. However, the
purification protocol needs to be optimized. For example, the Scl2.28 protein band is apparent in lane 7 of
SDS-PAGE of ligation combination 4 (6 hours expression) corresponding to the washing step of the purification.
This could be due to the buffer composition of the kit. The specific components of the buffer are not available in
the kit handbook.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/results-discussion/sds2c.png
      )

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/results-discussion/sds4.png
      )

# Transformation of E. coli auxotroph strain

E. coli JW0233 and JW2806 auxotroph strains were made competent using calcium chloride method. Competent cells were then transformed with plasmid 2C and 4 and plated on kanamycin and chloramphenicol agar plates. pUC19 was used as a positive control and plated on ampicillin agar plates. Growth was present in positive control plates indicating auxotroph strains were competent. Single colonies were not visible in Kan plates but was present in Cam plates.

![Collagen Structure](https://static.igem.wiki/teams/4812/wiki/competent.png
      )

# Future Steps

Purification needs to be repeated with revised buffer composition based on protocol available in literature [3] and expert consultation. Protocol is available on the experiments page. Once the protocol has been optimized, protein can be purified in larger quantities.

For residue-specific incorporation of hydroxyproline and hydroxylysine, growth curve optimization of auxotroph strains in M9 media containing various concentrations of proline and lysine respectively needs to be performed. From the growth curve analysis, the concentration at which the cells reach a plateau needs to be determined and indicates proline depletion. Subsequently, larger scale liquid cultures in M9 media can be cultured at the optimal concentration of proline or lysine and once culture reaches the desired OD, expression is induced using IPTG alongside the addition of hydroxyproline and hydroxylysine. We intend to compare two methods for residue-specific incorporation of Hyp ang Hyl, proline depletion as outlines here, and media shift as outlined by Breunig [5].

After producing the modified CLP, we aim to characterize its properties. Fourier-transform infrared spectroscopy (FTIR) will be used to analyze morphology and secondary structure, circular dichroism to investigate the triple-helical structure, and dynamic light scattering to assess self-assembly capabilities. We will also incorporate cell-adhesion domains through integrin binding sites and other functional domains to enhance interaction with cells. Cytotoxicity and biocompatibility tests will be performed to ensure safety for tissue engineering applications. We hope to explore the CLP's hydrogelation capabilities for 3D bioprinting through the addition of methacrylate groups for photo crosslinking or 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemical crosslinking.

The ability to design collagen-like protein is a salient feature made possible through synthetic biology. Tailoring the properties of collagen-like protein, like Scl2.28, has huge potential for enhancing tunability and improving the level of control over systems.

# References

1. Mohs, A., et al., Mechanism of Stabilization of a Bacterial Collagen Triple Helix in the Absence of Hydroxyproline\*. Journal of Biological Chemistry, 2007. 282(41): p. 29757-29765.
2. Yoshizumi, A., et al., Self‐association of Streptococcus pyogenes collagen‐like constructs into higher order structures. Protein Science, 2009. 18(6): p. 1241-1251.
3. Ilamaran, M., et al., A self-assembly and higher order structure forming triple helical protein as a novel biomaterial for cell proliferation. Biomaterials Science, 2019. 7(5): p. 2191-2199.
4. Baba, T., et al., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology, 2006. 2(1): p. 2006.0008.
5. Breunig, S.L. and D.A. Tirrell, Chapter Seventeen - Incorporation of proline analogs into recombinant proteins expressed in Escherichia coli, in Methods in Enzymology, E.J. Petersson, Editor. 2021, Academic Press. p. 545-571.

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