iGEM 2022 — Team tec-monterrey

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{% block title %}Notebook{% endblock %}
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# Week 1

* Since the delivery of the parts and distribution kit took longer
  than expected we started working with plasmids that were included in the iGem Distribution Kit from 2021,
  PSB1A3 and PSB1C3 both with RFP construct.
* We grew a culture with *E. coli* DH5α containing Pcomb 3x given by PhD Alejandro Gonzales to make a MiniPrep and have a stock of the plasmid for further use.
* We prepared several stocks of chemocompetent cells and saved them at -80°C in aliquots of 200 μL for all the future transformations procedures.

![miniprep](https://static.igem.wiki/teams/4506/wiki/miniprep.jpg)

**Figure 1.** Performing a miniprep of PSB1A3 and PSB1C3 from the iGem Distribution Kit 2021

# Week 2

* We transformed PSB1C3 and pBLUESCRIPT into DH10B and DH5α strains, respectively, to test the transformation efficiency of each cells.
* We grew a colony of the transformed pBLUESCRIPT given by CEM team and made a MiniPrep to save a stock of the plasmid and prepared a stock of the culture to be conserved at -80 °C in LB media with 13% glycerol.
* We transformed PSB1C3 and PSB1A3 into *E. coli* DH5α cells and obtained a few colonies and picked two of those colonies from each plasmid to miniprep, also we induced both constructs with IPTG to confirm functionality of the RFP construct and made stocks at -80 °C in LB media with 13% glycerol.
* An experiment to determine the optimal digestion time was performed with the enzymes PstI and EcoRI. In each sample we only changed the time using 1, 2 and 3 hours. The digestion products were run on an 1% agarose gel and the optimal time was 3 hours since this time gave us better band definition and less degraded DNA.

![miniprep](https://static.igem.wiki/teams/4506/wiki/redpellet.png)

**Figure 2.** Expression of RFP in the *E. coli* DH5α after the induction with IPTG

# Week 3

* Cultures of NEB 10 competent cells were grown overnight to propagate the strain and create new stocks of calcium competent cells.
* A culture of TG1 strain was also grown overnight to save an aliquot at -80°C.
* Digestions of PCB1C3 with the enzymes PstI and EcoRI were performed in order to test the performance of our enzymes against a stock provided by the academic department. It was determined that the enzymes owned by the team had higher efficiency with a 2 hour digestion and an enzyme inactivation at 80°C for 20 min. This did not explain the low quantity of digested plasmid product.
* Recovery of the digested plasmid from the agarose gel after electrophoresis was done using the Monarch gel purification kit, low concentrations of the plasmid DNA was recovered and quantified in the nanodrop.

![miniprep](https://static.igem.wiki/teams/4506/wiki/gel1.png)

**Figure 3.** Agarose gel 1% used to analyse the digestion products. From left to right: ladder of 1 kb, negative control, 1 hour digestion, 2 hours digestion, 3 hours digestion. All the digestions show degraded DNA and the 3 hour digestion was determined to be the optimal due to a better band definition and less degraded DNA.

# Week 4

* The first attempt of VCSM13 bacteriophage propagation was performed by infecting a 2mL TG1 culture using 2 μL of bacteriophage stock and incubating for 1 hour at 37°C and 250 rpm.
* The 2 mL of the infected culture were transferred to 2 falcon tubes with 15 mL of LB media each and incubated for 1 hour at 37°C and then kanamycin was added to both and incubated at 37°C overnight.
* The recovery of the M13 bacteriophages was performed by centrifugation and the supernatant was saved as stock.

# Week 5

* The parts we synthesized with IDT arrived and after resuspension we made a PCR to verify its integrity. The PCR product was run on a 1% agarose gel.
* We started to prepare all the necessary for the cloning of the gBlocks. The digestion of the backbones was performed and we noticed big amounts of degraded DNA were in the bottom of every lane of the electrophoresis gel. At this point we were obtaining high recovery yields from the MiniPreps (2000-5000 ng/μL), so we expected a good amount of digested backbones.
* To solve this problem we performed each digestion by triplicate to concentrate and get a higher amount of digested backbone, however concentrations were low, less than 10 ng/ul.

![miniprep](https://static.igem.wiki/teams/4506/wiki/plasmiddegradated.jpeg)

**Figure 4.** Agarose gel 1% used to analyse the digestion products. From left to right: ladder of 1 kb, PSB1A3 by triplicate, PSB1C3 by triplicate, Pcomb3x by triplicate. Note the degraded DNA in each digestion reaction

PCRs at different annealing temperatures (a range of 55°C to 70°C) to identify the optimum temperature of Axtl primers were performed. 61°C was the best annealing temperature for this set of primers

# Week 6

* We digested the parts and proceeded with a fast ligation (10 min at room temperature) for the first cloning attempt with the IDT gBlocks.
* We transformed the ligations and had not successful results.
* Twist parts arrived, after they were held by the custom house for two months at room temperature. We amplified the gBlocks by the first time and identified that the parts presented considerable degradation, also we could not amplify from PCR products, indicating poor DNA integrity with the parts.

![miniprep](https://static.igem.wiki/teams/4506/wiki/gelpcrparts.png)

**Figure 5.** Agarose gel 1% showing the degradation of the Twist parts

# Week 7

* To avoid the degraded plasmidic DNA, and try to have more digested plasmids, we changed the minipreps solutions with freshly made ones and also tested other ones borrowed to make a comparison between the quality of the recovered DNA.
* Even with this strategy we still presented a lot of degraded DNA so we decided to use a borrowed kit to make the MiniPreps, we obtained a lower recovery yield (250-450 ng/μL), but with a better quality, since no degraded DNA was identified by 1% electrophoresis gel.
* Digestions with the kit minipreps were performed and we obtained acceptable amounts that we could recover from the gel (9-34ng/μL), for this we concentrated with magnetic beads a single miniprep of each plasmid in a lower volume.

![miniprep](https://static.igem.wiki/teams/4506/wiki/concentratedplasmids.jpeg)

**Figure 6.** Agarose gel 1% used to analyse the digestion products. From left to right: ladder of 1 kb, PSB1A3 by duplicate using 15 enzyme units, PSB1C3 by duplicate using 15 enzyme units, Pcomb3x by duplicate using 15 enzyme units, PSB1A3 concentrated, PSB1C3 concentrated, Pcomb3X concentrated. Note the supperior yield and lack of inespecific activity on the last 3 reaction

# Week 8

* With the new digested backbones using MiniPreps from kit, the second clonation attempt was done using fast ligation (10 min at room temperature), and a high molar ratio of approximately 5:1 insert-backbone.
* The third clonation attempt took place, with new digested parts and using longer ligation times (16°C overnight) and a higher molar ratio approximately 7:1 insert-backbone. Two ligation products provided colonies and were tested, giving a positive colony PCR confirming the insert was successfully cloned. The need for higher insert:vector yield could be explained by the unavility of the parts to be accessible for ligations as consequence of the degradation process
* Digestions with the kit minipreps were performed and we obtained acceptable amounts that we could recover from the gel (9-34ng/μL), for this we concentrated with magnetic beads a single miniprep of each plasmid in a lower volume.
* Digestions with the kit minipreps were performed and we obtained acceptable amounts that we could recover from the gel (9-34ng/μL), for this we concentrated with magnetic beads a single miniprep of each plasmid in a lower volume.

![miniprep](https://static.igem.wiki/teams/4506/wiki/anewhope.jpeg)

**Figure 7.** Agarose gel 1% used to analyse the colony PCR products. From left to right: Control, Colony 1 PSB1A3+FinalCamRNA, Colony 2 PSB1A3+FinalCamRNA, Colony 1 PSB1A3+ManualCamRNA , Colony 2 PSB1A3+ManualCamRNA

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##### Section Index

## Software

## Wet Lab

[Sections](#sections)

## Software

## Wet Lab

## Software

Since the actual protocols state that the sRNA design must consider the first 24 nucleotides on the target mRNA without studying thermodynamic parameters, we had to design our first constructs following this methodology. In order to improve sRNA functionality we decided to create a python-based pipeline capable of designing sRNAs targeting an entire gene and then applying a scoring criteria following literature parameters. Since the mechanisms underlying sRNA.mediated gene regulations remain unclear to date, we have developed a neural network algorithm (trained with information of natural sRNA:mRNA pairs) in order to predict the regulation behavior (upregulation or downregulation) of the previously created sRNAs. It is important to note that since all of the created sRNAs have a Hfq-binding domain, the expected effect is downregulation, however since the effects of synthetic sRNAs have not been well characterized when the target regions are located far downstream, we decided to create the neural network model.

![srnamachinelearning](https://static.igem.wiki/teams/4506/wiki/srna-machine-learning.png)

Image 1. Workflow diagram for design and improve of sRNA constructs for gene downregulation

## Wet Lab

When developing our project we went through the Design→Build→Test→Learn cycle several times. As part of our original plan to be performed in the lab, we were considering using the M13KO7 helper phagemid to construct the non-modificated phages and test the silencing efficiency of the sRNAs in *E. coli TG1*. Nevertheless, Mexican suppliers of the M13KO7 did not have an available stock to be sold and to be arrived in a short period of time. We tried with an international supplier but a significant difference in price was notable, and we wanted to avoid all the shipping problems that would have been involved in this specific scenario, as we have been through with parts and reagents ordered by an international supplier. As part of the interaction with Ph.D. Alejandro Gonzalez, he offered us an aliquot of the VCSM13 helper phage that he had in our college laboratories readily available for us. VCSM13 helper phage is a derivative of M13KO7, and both are a good option when selecting a helper phage, nevertheless, before VCSM13 was offered to us M13KO7 was easier to access as is the one Mexican suppliers usually work with, but after Ph.D. Alejandro Gonzalez's offer, changing M13O7 for VCSM13 was an excellent option to optimize our two less available resources, time and money.

Our original plan also considered using a construct of the chimeric GP3 protein (BBa\_4506000) and cloning it into PSB1A3 to perform the phage display in *P. aeruginosa*. Nevertheless, we presented some errors at the time our parts were evaluated by IDT due to its complexity to be synthetized, as a consequence we had to make some modifications to the GP3 chimeric protein for the phage display experiment. Ph.D. Alejandro Gonzalez helped us with this by kindly offering an aliquot of *E. coli* DH5α containing Pcomb3XTT backbone, which he used to successfully perform phage display in M13 bacteriophages, by changing also the N terminal of the P3 M13 protein. With this alternative we had to create a new derivative of the GP3 chimeric construct containing only the N terminal of the P3 protein of the PF1 phage, Pcomb3XTT had already the F1 replication origin and accessible cut sites to digest and then clone the insert, thus it was an excellent substitute to the original plan.

![cassettedesign](https://static.igem.wiki/teams/4506/wiki/codigo-engineering.png)

Image 2. Design of the cassette harboring the chimeric construct OmpA+ N term -Pfl

![gblockflowchart](https://static.igem.wiki/teams/4506/wiki/flowchart-gblock.png)

Image 3. Flowchart of the gBlock construction strategy for the synthesis of our chimeric protein

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##### Section Index

## PCR

## PCR Purification

## Electrophoresis

## Gel Purification

## Digestion

## Double Digestion

## Competent Cells Preparation

## Transformation

## Miniprep

## Colony PCR

## IPTG Induction

## Bead Purification

## Phage propagation

## Phage titration

## Phage Purification

[Sections](#sections)

## PCR

## PCR Purification

## Electrophoresis

## DNA from gel

## Digestion

## Double digestion

## Competent Cells

## Transformation

## Miniprep

## Colony PCR

## IPTG Induction

## Bead Purification

## Phage propagation

## Phage titration

## Phage Purification

## PCR

We recommend assembling all reaction components on ice and quickly transferring the reactions to a
thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to
use.

### Materials

* Q5 High-Fidelity 2X Master Mix
* 10 µM Forward Primer
* 10 µM Reverse Primer
* DNA Template
* H2O (Nuclease-free)

### Procedure

* Add Q5 High-Fidelity 2X Master Mix to a PCR tube.
* Add each Primer to a final concentration of 500 nM.
* Add up to 10 ng of Template DNA.
* Add Nuclease-Free Water up to final reaction volume (2 times the volume of master mix).
* Gently mix the reaction.
  + Alternatively if doing several reactions and using the same amount of template for each one, a
    pre-mixture of the remaining reagents can be prepared and then distribute the amount of mix in
    separate tubes prior to adding the template DNA.
* Collect all liquid to the bottom of the tube by a quick spin if necessary
* Quickly transfer PCR tubes to a thermocycler previously set to the following configuration:

  | Step | Temperature | Time |
  | --- | --- | --- |
  | Initial denaturation | 98°C | 30 Seg |
  |  | 98°C | 10 Seg |
  | 25 to 35 Cycles\* | 60°C | 20 Seg |
  |  | 72°C | 25 Seg |
  | Final extension | 72°C | 2 Minutes |
  | Hold | 4°C | ∞ |

  \*Use less than 20 cycles if amplifying pure synthesized parts

## PCR product purification

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A
starting sample volume of 20–100 µL is recommended. For smaller samples, TE can be used to adjust the
volume to the recommended volume range.
**Centrifugation should be carried out at 16,000 x g (~13K RPM in a typical microcentrifuge) in a
standard laboratory microcentrifuge at room temperature.**

### Materials

* Monarch® DNA Cleanup Columns (5 μg)
* Monarch Collection Tubes (2mL)
* Monarch® DNA Wash Buffer
* Monarch® DNA Cleanup Binding Buffer
* Monarch® DNA Elution Buffer or Nuclease-Free Water
* Ethanol (≥ 95%)

### Before you begin

Add 20 mL of ethanol to the Monarch DNA Wash Buffer (4 volumes of ≥ 95% ethanol per volume of Monarch
DNA Wash Buffer).

### Procedure

* Dilute sample with DNA Cleanup Binding Buffer according to the table:

  | Sample type | Binding buffer:Sample ratio | Example |
  | --- | --- | --- |
  | dsDNA > 2 kb (plasmids, gDNA) | 2:1 | 200 µL Buffer: 100 µL sample |
  | **dsDNA < 2 kb (some amplicons, fragments)** | **5:1** | **500 µL Buffer:100 µL sample** |
  | ssDNA > 200 nt | 7:1 | 700 µL Buffer: 100 µL sample |
* Mix well by pipetting up and down or flicking the tube. **Do not vortex.** A starting sample
  volume of 20–100µL is recommended.
* Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute,
  then discard flow-through.
* Re-insert column into collection tube. Add 200µL DNA Wash Buffer and spin for 1 minute. Discarding
  flow-through is optional.
* Repeat previous step.
* Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does
  not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of
  salt and ethanol are not carried over to the next step.
* Add ≥ 6 µL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1
  minute to elute DNA.
  + **Note:** Typical elution volumes are 6–20 µL. Nuclease-free water (pH 7–8.5) can also be
    used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is
    used, but the DNA will be less concentrated

## Electrophoresis

QUICKSILVER™ TAE Buffer Powder and SmartGlow PS (E4500) are used for this protocol. Protocol will
change with other products.

### Materials

* QUICKSILVER™ TAE Buffer Powder
* Distilled water
* SmartGlow PS
* Agarose
* SmartCheck Ladder

### Procedure

TAE Buffer (To make 1L of buffer)* Add contents of buffer pouch to a graduated cylinder or beaker.
* Slowly add distilled water to 1 liter.
* Mix well until all granules are dissolved.
* Add additional water to bring the volume to 1L, if necessary.

Agarose Gel (1%)* Add 0.5g of agarose to 50 mL of TAE buffer and microwave to dissolve.
* Add 2.5 µL of SmartGlow and mix.
* Pour gel into gel tray.
* Let it sit until solidified and place into the tank.
* Add TAE buffer into the tank until the gel is covered.

+ **NOTE:** For better results, 5uL of SmartGlow can be added per 100mL of TAE running buffer.

Sample preparation and gel observation* Add 5-10 µL of Ladder
* Mix X6 loading dye with samples and DNA markers at a 1:5 (dye to sample) dilution.
* Run at 100V for 45 to 60min.
* View the gel using a UV or blue light.

## DNA from gel purification

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM). Note that the columns hold
up to 800uL.

### Materials

* Monarch® DNA Cleanup Columns (5 μg)
* Monarch Collection Tubes (2mL)
* Monarch® DNA Wash Buffer
* Monarch® Gel Dissolving Buffer
* Monarch® DNA Elution Buffer or Nuclease-Free Water
* Ethanol (≥ 95%)

### Before You Begin

Add 20 mL of ethanol to the Monarch DNA Wash Buffer (4 volumes of ≥ 95% ethanol per volume of Monarch
DNA Wash Buffer).

### Procedure

* Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other
  clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 mL microcentrifuge tube
  and weigh the gel slice.
* Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel slice (e.g., 400 µL buffer
  per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving
  Buffer to 3 or 3.5 volumes to minimize the guanidine salt present.
* Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is
  completely dissolved (generally 5–10 minutes).
* Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then
  discard flow-through.
* Re-insert column into collection tube. Add 200 µL DNA Wash Buffer and spin for 1 minute. Discarding
  flow-through is optional.
* Repeat previous step.
* Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does
  not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of
  salt and ethanol are not carried over to the next step.
* Add ≥ 6 µL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1
  minute to elute DNA.
  + **Note:** Typical elution volumes are 6–20 µL. Nuclease-free water (pH 7–8.5) can also be
    used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is
    used, but the DNA will be less concentrated

## Digestion

* Set up reaction as follows:

  | Component | 20 µL REACTION |
  | --- | --- |
  | DNA | >1 µg |
  | 10X rCutSmart Buffer | 2 µL (1X) |
  | EcoRI-HF | 0.5 µL (10 units) |
  | Nuclease-free Water | to 20 µL |
* Incubate at 37°C for at least 2 hours (EcoRI maintains its activity for at least 4-8 hours).
  + For convenience, 0.5µL is specified; adjust as needed. In general, 5–10 units of enzyme per µg
    DNA, and 10–20 units for genomic DNA in a 1 hour digest is recommended.
  + Reaction volumes can be linearly adjusted up or down, however enzyme volume should not exceed
    10% of the total reaction volume to prevent star activity due to excess glycerol.
* Optional: Heat inactivate at 65°C for 20 min.
  + Heat inactivation is recommended if you plan to continue to another step in the workflow without
    a DNA purification step.

## Double-digestion

* Set up reaction as follows:

  | Component | 20 µL REACTION |
  | --- | --- |
  | DNA | >1 µg |
  | 10X NEBuffer 2.1 | 2 µL (1X) |
  | EcoRI-HF | 0.5 µL (10 units) |
  | PstI | 0.75 µL (15 units) |
  | Nuclease-free Water | to 20 µL |

  **Note:** PstI has only 75% of activity in NEBuffer 2.1 and EcoRI has 100%, PstI is added in
  a ratio of 1.5:1 to EcoRI in order to compensate for the difference.
* Incubate at 37°C for at least 2 hours (EcoRI maintains its activity for at least 4-8 hours and PstI
  for at least 2-4 hours).
  + For convenience, 0.5µL is specified; adjust as needed. In general, 5–10 units of enzyme per µg
    DNA, and 10–20 units for genomic DNA in a 1 hour digest is recommended.
  + Reaction volumes can be linearly adjusted up or down, however enzyme volume should not exceed
    10% of the total reaction volume to prevent star activity due to excess glycerol.
* Optional: Heat inactivate at 80°C for 20 min.
  + Heat inactivation is recommended if you plan to continue to another step in the workflow without
    a DNA purification step.

## Competent Cells Preparation

### Materials

* SOB
  + 0.5% (w/v) yeast extract
  + 2% (w/v) tryptone
  + 10 mM NaCl
  + 2.5 mM KCl
  + 20 mM MgSO4
* CCMB80 buffer
  + 10 mM KOAc pH 7.0
  + 80 mM CaCl2
  + 20 mM MnCl2
  + 10 mM MgCl2
  + 10% glycerol
  + Adjust pH DOWN to 6.4 with 0.1N HCl if necessary
    - Slight dark precipitate does not affect its function, but makes the next step more
      difficult.
  + Sterile filter and store at 4°C

### Procedure

* Inoculate 250 mL of SOB medium with 1 mL vial of seed stock and grow at 20°C to an OD600nm of 0.3.
  + This takes approximately 16 hours.
  + Room temperature will work. You can adjust this temperature somewhat to fit your schedule.
  + **Aim for lower, not higher OD if you can't hit this mark.**
* Pre-chill in ice as many centrifuge tubes as needed.
* Transfer the culture to the chilled centrifuge tubes.
* Centrifuge at 3000g at 4°C for 10 minutes.
* Discard supernatant.
* Add 40mL of ice cold CCMB80 buffer first to resuspend the cells.
* When cells are in suspension, add another 40mL of CCMB80 buffer for a total of 80mL.
* Pipet buffer against the wall of the centrifuge bottle to resuspend cells.
  + Avoid pipetting directly into cell pellets.
* Incubate on ice for 20 minutes.
* Centrifuge at 3000G at 4°C for 10 minutes.
* Discard supernatant.
* Resuspend cell pellet in 10 mL of ice cold CCMB80 buffer.
  + If using multiple centrifuge tubes, combine the cells post-resuspension.
* Incubate on ice for 20 minutes. Prepare for aliquoting
* Aliquot 200 µL into chilled PCR tubes.
* Store at -80°C indefinitely.
**Note:**
Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency and
thus leftover cells must be discarded.

## Transformation

### Procedure

* Thaw a tube of competent E. coli cells on ice until the last ice crystals disappear. Mix gently and
  carefully pipette 50 µL of cells into a 1.5 mL microcentrifuge tube on ice.
* Add 1-5 µL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5
  times to mix cells and DNA. **Do not vortex.**
* Place the mixture on ice for 30 minutes. **Do not mix.**
* Heat shock at exactly 42°C for **exactly 30 seconds. Do not mix.**
* Place on ice for 5 minutes. **Do not mix.**
* Pipette 950 µL of room temperature SOC into the mixture.
* Place at 37°C for 60 minutes shaking at 250 RPM.
* Prepare LB Agar selection plates with the adequate antibiotic.
* Spread 100 µL of the culture onto a selection plate and incubate overnight at 37°C.
  + Dilute prior to plating if necessary.

## Miniprep

Isolation and propagation of DNA molecules are essential features of recombinant DNA technologies. To
this purpose, DNA molecules are copied (propagated) using the replication machinery of its host. A
plasmid or a vector is a circular DNA molecule that contains the information necessary for its
replication inside a host. Plasmids and vectors contain antibiotic resistance genes allowing selection
of bacteria hosting the plasmid of interest when a population of bacteria is cultured in the presence
of the antibiotic. Plasmids and vectors also contain specific DNA sequences recognized by restriction
enzymes allowing the insertion of DNA fragments through the combination of a host plasmid cut by a set
of restriction enzymes and a DNA fragment with compatible ends in a reaction mediated by a ligase. The
newly ligated plasmid containing the DNA insert will be transformed into a host to replicate and
propagate itself. This host-assisted heterologous-DNA fragment replication process is known as
cloning.

### Materials

* Solution I
  + Tris HCl 25 mM
  + EDTA 10 mM
* Solution II
  + NaOH 0.2 N
  + SDS 41.9 mM
    - SDS must be dissolved carefully.
* Solution III
  + Potassium acetate 3 M
    - Must be previously dissolved in water.
  + Acetic Acid 2 M
* 1.5 mL microcentrifuge tubes
* Isopropanol
* Ethanol 70%
* Nuclease-Free Water
* RNAse

### Before you begin

Add 10 mL of LB media to a 50mL tube with the corresponding antibiotic, inoculate with a single colony
from a plate. **All cultures were incubated overnight at 37ºC and shaken at 250 rpm.**

### Procedure

* Centrifuge for 5 min at 5000xg.
* Discard supernatant.
* Add 200 μL of Solution I and 1 μL of RNAse.
* Resuspend until the pellet is completely solubilized.
* Incubate 5 min at room temperature.
* Add 200 μL of Solution II.
* Mix by inversion.
* Incubate 5 min at room temperature.
* Add 200 μL of Solution III.
* IMix by inversion.
* Incubate 10 min at -20ºC.
* Centrifuge for 10 min at 16000xg (14,000 RPM).
* Transfer supernatant to a new 1.5 mL tube.
  + Avoid disturbing/touching white precipitate.
* Add 500 μL of isopropanol.
* Centrifuge for 10 min at 16000xg (14,000 RPM).
* Discard supernatant.
* Add 1 mL of ethanol 70%.
* Mix by vortexing.
* Centrifuge for 5 min at 16000xg (14,000 RPM).
* Discard supernatant.
* Dry tubes with open cap at 37ºC for 45 min.
* Add 50 μL of Nuclease-Free water.
* Mix by vortexing.
* Storage at 4ºC for short periods of time (days) or -20ºC indefinitely.

## Colony PCR

We recommend assembling all reaction components on ice and quickly transferring the reactions to a
thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to
use.

### Materials

* PCR tubes
* Nuclease-Free water
* Sterile loop

### Procedure

* Add 10-20 µL of Nuclease-Free water into a PCR tube.
* Pick a single colony with a sterile loop.
* Inoculate the water with the loop, making sure most bacteria ends in the water.
  + The water must become somewhat cloudy.
* Incubate at 95°C for 10 minutes.
  + This could be easily done in a thermocycler.
* Perform a usual [PCR procedure](https://2021.igem.org/Team%3ATec-Monterrey/Experiments#pcr)
  using 1-2 µL of the lysed bacteria as template.

## IPTG Induction

IPTG or Isopropyl β-D-1-thiogalactopyranoside is a chemical reagent mimicking allolactose, which removes a repressor from the lac operon to induce gene expression, since our promoters work by the interaction of the lac repressor IPTG Induction allow us to express our protein of interest.

### Procedure

* Transform expression plasmid into *E.coli* BL21(DE3).
* Plate on antibiotic selection plates and incubate overnight at 37°C.
* Resuspend a single colony in 10 mL liquid culture with proper antibiotic.
* Incubate at 37°C until OD600 reaches 0.4–0.8.
* Induce with 10 µL of a 1 M stock of IPTG (final concentration of 1 mM) and induce for 2-4 hours at
  37°C.
* Analyze by method of choice.

## Purification by SPRI beads technology

The magnetic microparticles with the SPRI (solid-phase reversible immobilization) technology are a high-throughput DNA and RNA purification strategy. AMPure XP utilizes an optimized buffer to selectively bind DNA fragments 100 bp and larger to paramagnetic beads. Excess primers, nucleotides, salts, and enzymes can be removed using a simple washing procedure. The result is a more purified and/or concentrated sample.

### Materials

* AMPure XP beads
* Fresh 80% ethanol
* Nuclease free water

### Procedure

* Determine whether or not a plate transfer is necessary.
* Shake the AMPure XP bottle to resuspend any magnetic particles that may have settled. Then add AMPure XP according to the sample reaction volume.
* Mix reagent and sample thoroughly by pipette
  mixing 10 times. Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery. The color of the mixture should appear homogeneous after mixing.
* Mix reagent and sample thoroughly by pipette
  mixing 10 times. Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery. The color of the mixture should appear homogeneous after mixing.
* Place the reaction plate onto a magnet plate for 2 minutes to separate beads from the solution. Wait for the solution to clear before proceeding to the next step.
* With the reaction situated on the magnet, aspirate and discard the cleared solution from the reaction plate and discard.
* Dispense 200 μL of 80% ethanol to each well of the reaction plate and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard.
* Repeat the last step for a total of two washes.
* Remove the reaction plate from the magnet plate, and then add the necessary volume (10 - 40 µL)of NFW to each well of the reaction plate and pipette mix 10 times. Incubate for 2 minutes.
* Place the reaction plate onto a magnet plate for 1 minute to separate beads from the solution.
* Transfer the eluate to a new plate.

## M13 bacteriophage propagation and preparation

Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension, this propagation is necessary in order to perform infection protocols and phage display procedures.

### Procedure

* Incubate a 2 mL culture of E. coli TG1 until a log phase is reached, this allows for a more efficient infection.
* Add 2 μL of VCSM13 bacteriophage stock and incubate at 37°C and 250 rpm for 1 hour.
* Transfer the 2 mL infected culture to 2 falcon tubes each with 15 mL LB media.
* Incubate at 37°C and 250 rpm for 1 hour.
* Add kanamycin to a final concentration of 35 μg/mL to each falcon tube.
* Incubate overnight at 37°C and 250 rpm.
* Centrifugate the tubes at 2500g for 15 minutes.
* Transfer the supernatants to new falcon tubes and incubate in a water bath at 70°C for 20 minutes.
* Centrifugate the tubes at 2500g for 15 minutes and transfer the supernatants to new falcon tubes.
* Store at 4°C.

## M13 bacteriophage titer determination

### Procedure

* Inoculate 2 mL of LB medium with 2 μL of E. coli TG1 at 37°C and 250 rpm for 1 hour.
* Prepare 10-7, 10-8 and 10-9 dilutions of the bacteriophage preparation in LB medium.
* Use 1 μL of the dilutions to infect 50 μL of the TG1 culture and incubate for 15 minutes at room temperature.
* Add 3 mL of LB medium and pour into LB agar plates andIncubate overnight at 37°C
* Determine the titer from the number of plaques. 50 plaques derived from the 10-8 solution corresponds to a titer of 5x1012 plaque-forming units (pfu) per mL. The titer should be in the range of 1012 to 1013 pfu/mL.
* The titer tends to decrease with time and the bacteriophage preparation is stable for many months at 4°C.

## M13 bacteriophage purification

### Procedure

* Incubate the bacteriophage preparation with PEG 6000 at 4°C with continuous agitation for 90 minutes.
* Centrifugate at 10000 rpm for 10 minutes and discard the supernatant.
* Resuspend the pellet in deionised water.
* Lower the pH to 4 using 5M HCl.
* Centrifugate at 15000 rpm for 5 minutes and discard the supernatant.
* Resuspend the pellet in deionised water and centrifugate again at 15000 rpm for 5 minutes.
* Resuspend the pellet in deionised water and store at -20°C until ready to use.

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##### Section Index

## Wet Lab

## Software

## Microfluidics

## Nebulizer

[Sections](#sections)

## Wetlab

## Software

## Microfluidics

## Nebulizer

## Wet lab results

The cloning process of our parts faced us with important challenges that needed to be solved at the last minute, since most of our parts were delivered in bad conditions, being held in the Mexican customs at room temperature and opened. We noticed that we could amplify the parts when the DNA template was from the resuspended stock, but we could not use the PCR product as a new template since no amplification was obtained from this, even when changing the primers, polymerase and reaction conditions (annealing temperature). This last problem was mainly attributed to the bad shipping conditions that caused the 96 well plate containing our parts to arrive open and to be held at room temperature for almost two months. As a consequence our parts presented considerable degradation that we could notice since the first amplification was made.

![miniprep](https://static.igem.wiki/teams/4506/wiki/gelpcrparts.png)

**Figure 1.**  Initial PCR of the Twist parts recently opened

However, by using increasing amounts of digested DNA for the ligation process we were able to obtain a successful ligation. After overnight growth we performed a colony PCR that showed a band in the expected size, as well as showing the presence of RFP reporter gene after propagation of those bacteria in LB medium provided with the selection marker. Unfortunately we were not able to characterize the effectiveness of the sRNAs since time was a limiting factor.

![miniprep](https://static.igem.wiki/teams/4506/wiki/anewhope.jpeg)

**Figure 2.** Agarose gel 1% used to analyse the colony PCR products. From left to right: Control, Colony 1 PSB1A3+FinalCamRNA, Colony 2 PSB1A3+FinalCamRNA, Colony 1 PSB1A3+ManualCamRNA , Colony 2 PSB1A3+ManualCamRNA

One the other hand, we had the chance to characterize the Axtl primers, these primers were recovered from the cytochrome c oxidase subunit I (COX1) gene of Ambystoma mexicanum (axolotl).

| Primer | Sequence(3' to 5') | Lenght (bp) |
| --- | --- | --- |
| Axtl Fw AGCTGGAACGGGATGAACTG | 20 | |
| Axtl Rev CGTCGTGGTATTCCGGCTAA | 20 | |

We amplified a sample with five different annealing temperatures between a range of 55°C to 70°C to identify the optimum for the Axtl primers. From this experiment, 61°C showed to be the optimum temperature. However in our first attempt we observed similar amplification levels in all temperatures, we assume this result was a product of the high sensitivity of our polymerase Primer Star Max; to resolve this we performed a second experiment using Thermo Fisher Taq Polymerase and obtained similar results, confirming this was a product of a good primer design that showed good annealing stability.

![optm](https://static.igem.wiki/teams/4506/wiki/op-tm.png)

**Figure 1.** Agarose gel 1% used to analyse optimal annealing temperature of PCR products with Axtl primers. From left to right: 1kb Ladder, 55 °c, 58 °C, 61 °C, 64 °C, 67 °C

We plan on performing further work to expand on our solution. Despite the limitations encountered in the lab, we were able to generate other results that go beyond the lab and use mathematical and systematic approaches to expand on our project.

## Software results and conclusions

As part of the mission of the project, our team designed a dataset of small RNAs using our python pipeline for targeting cloramphenicol-acetyl transferase and penicillin beta-lactamase genes in bacteria. The profile of the score vs the binding site of the created sRNAs is on figure 2.

![miniprep](https://static.igem.wiki/teams/4506/wiki/graphicsresults.png)

**Figure 2.** Results of the sRNAs analized by sRNA Designer

As noted on figure 2, higher scores for sRNA hybridization efficiency can be achieved if the target region is located downstream of the start codon. Since this is the first attempt to merge both scores 1 and 2, our team decided to select three sRNAs with improved characteristics per gene: one with the higher score 1 value, a second one with a higher S2 value and the last one with higher final score (S1+S2). It is important to note that we have also considered the sRNAs made by basepairing to the first 24 nts as a control, since the actual protocols state that a sRNA must pair to these site.

![miniprep](https://static.igem.wiki/teams/4506/wiki/tablamodelo.png)

Despite the fact that scores are normalized, the higher reported scores are not equal to 1 (S1 and 2) or 2 (final score) since the selection criteria used in the present project is only to choose those sRNAs inside a 100-nt downstream gene start region, as explained on our design.

However, after a suscessfull neural-network based model incorporation into our software (see model and software) we were able to calculate the best sRNA for downregulate the cloramphenicol-acetyl transferase gene in E. coli regardless the basepairing site previosuly mentioned limitations. It is important to note that since our neural network model was trained with reported sRNA:mRNA pairs retrieved from literature (in which the basepairng position was present) we consider that we can incorporate any sRNA that give suscessful results according to our modelling regardless the basepairing site

![miniprep](https://static.igem.wiki/teams/4506/wiki/finalfinalfinal.png)

**Table 2.** sRNAs created by our software in order to downregulate cloramphenicol-acetyl transferase gene in bacteria. This ones were created considering the incorporation of our neural network model. For more details about the titles see model and software pages

## Microfluidic results

The proof of concept related to the microfluidic encapsulation of the bacteriophages relies on the principle of the microfluidic coaxial device that is proposed to produce the liposome-phage. It is based on the injection of an organic phase into an aqueous phase using a constant and controlled flow rate to induce a laminar flow. In literature, this type of device has already been used for this purpose. The results showed the production of unilamellar liposomes containing each one or two phages. It has also been observed that the proportion of charged liposomes is superior when compared to the proportion of empty liposomes, proving a high rate of encapsulation [2].

The diffusion of molecular species at the interface of the organic phase with the aqueous phase induces the formation of liposomes by auto assembly due to the high polar environment created when the organic phase encounters the aqueous phase [2,3]. In COMSOL, the channels were constructed by extruding work planes containing their transversal geometry. The measures of the device were the same as the ones reported by Vladisavljević, G. T et al. [1] in order to compare the results of the simulation. The inner channel has a round shape (0.58 and 1 mm for the inner and outer diameter respectively) and the outer channel has a squared shape (1mm and 1.2 mm for the inner and outer side length). At the end of the inner channel a cone-shape structure with an inner diameter of 209 m was constructed. From the materials database of COMSOL, glass borosilicate was assigned to the domains and frontiers of the channels and water was used for both inner and outer channel’s fluids.

In order to obtain the velocity field and the flow line of fluids in the channels, the physic module of laminar flow was used. Two entries corresponding to the inner and outer channels were defined; a flow rate of 1.610-10 was used for the inner channel during all simulations and the flow rate of the outer channel was 0.6, 3 and 4.8 ml/h for each of the three simulations. For the boundaries, no slip condition was assigned to the channels of the walls since they are solid. For the computation, a tetrahedral mesh was used because we constructed a 3D model.

First, a stationary study was modeled in order to obtain the velocity field and the flow lines. In the model, the maximum velocity is reached at the center of the outlet of the inner channel as reported in the simulation made by Vladisavljević, G. T et al. [1]. The velocity at the walls is zero as shown in the velocity field of our three simulations.

The velocity of fluids in the channels depends on their cross-sectional area; they are inversely proportional. At the end of the inner channel, the velocity is higher than at the entrance and it decreases when the fluid enters the outer channel as a result of the augmentation of the cross-sectional area and the drag force from the aqueous phase (see Figure 1, 2 and 3). The results of our simulation indicate that as the flow rate of the aqueous phase increases; the velocity at the center of the outer channel also increases.

![microfluidicmodel](https://static.igem.wiki/teams/4506/wiki/modelo-microfluidico1.png)

Figure 1. Velocity field computed for flow rates of 0.6 ml/h for both the organic and aqueous phases.

![microfluidicmodel](https://static.igem.wiki/teams/4506/wiki/modelo-microfluidico2.png)

Figure 2. Velocity field computed for flow rates of 0.6 ml/h and 3 ml/h for the organic and aqueous phases respectively.

![microfluidicmodel](https://static.igem.wiki/teams/4506/wiki/modelo-microfluidico3.png)

Figure 3. Velocity field computed for flow rates of 0.6 ml/h and 4.8 ml/h for the organic and aqueous phases respectively.

When comparing the values of the maximum velocity in the inner channel obtained by our simulation using a static flow rate for the organic phase and a variable flow rate for the aqueous phase, the values are comparable in magnitude; 910-3m/s and 810-3m/s for the value reported in literature and the one obtained with our three simulations respectively. However, when the flow rates of aqueous phase are increased, there is a difference in the maximum velocity reached at the outer channel, for example for flow rates of 0.6 ml/h and 4.8 ml/h for the organic and aqueous phase respectively, the maximum velocity at the outer channel is three times bigger in our simulation (410-3m/s and 1210-3 for the literature and the model respectively). This mismatch could be explained by the difference in values of the properties of fluids used for each simulation; neither the type of fluid nor the density used for the simulations are specified by Vladisavljević, G. T et al. [1]. In the future, the density of the organic mix and the phage solution need to be determined to achieve more accurate results with our simulation.

Finally, the particle tracing module was used in order to observe the trajectory of the bacteriophages. Solid particles simulating the phages contained in the aqueous phase are released from aleatory positions at the inlet of the outer channel in the model in COMSOL. One particle was released each 0.1 s to observe its behavior. As shown in the animation, the velocity reached by the phages is the same as the one reported in the velocity field. The flow lines respect the boundaries of the model indicating its correct functioning.

In the future, we propose to fabricate the device in order to use microscopy to m onitor in real time the behavior of the fluids in the system. If it works correctly, a jet must be observed at the end of the inner channel; when increasing the flow rate of the aqueous phase, the jet must be shorter and thinner. If the size of the jet is closer to the diameter of the inner channel, a better mixing will be achieved. A better mixing leads to produce smaller liposomes according to Vladisavljević, G. T et al. [1]. It has been stated that the mixing grade depends on the flow rates and properties of the fluids and on the geometry of the channels, specifically on the diameter of the exit of the inner channel [1,2]. We expect our model to produce liposome-phage complex correctly because the behavior of our simulations is comparable to the one reported in literature.

![microfluidicmodel](https://static.igem.wiki/teams/4506/wiki/microfluidopt.gif)

## Nebulizer prototype

In order to make the delivery of the liposome-phage complex correctly we created a model prototype using electronics and 3D printing. The prototype made was based on the design of a membrane nebulizer "Omron NE-022" [3], designing a model that simulated its operation using an ultrasonic nebulizer, in light of a vibrating membrane that would be implemented in the real device. The base or box of the device was made by 3D printing, while SLA/DLP printing for the dispenser, as well as FDM printing for the rest of the system, On the other hand, for the electronics, we adapt the design of “Nano mist” [2] modifying it to extend the button to the base of the device and changing the Lipo battery for a Li-ion MS18650 battery.

**Figure 4.** Video of the nebulizer prototype.

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