iGEM 2021 — Team Tec-Monterrey

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# Team:Tec-Monterrey/Notebook

[![Main logo](https://static.igem.org/mediawiki/2021/3/39/T--Tec-Monterrey--logo-diagnosgene-blanco-stretch.svg)](/Team%3ATec-Monterrey)

* PROJECT

  [Description](/Team%3ATec-Monterrey/Description)
  [Design](/Team%3ATec-Monterrey/Design)
  [Notebook](/Team%3ATec-Monterrey/Notebook)
  [Experiments](/Team%3ATec-Monterrey/Experiments)
  [Proof of Concept](/Team%3ATec-Monterrey/Proof_Of_Concept)
  [Engineering Success](/Team%3ATec-Monterrey/Engineering)
  [Implementation](/Team%3ATec-Monterrey/Implementation)
  [Safety](/Team%3ATec-Monterrey/Safety)
  [Contribution](/Team%3ATec-Monterrey/Contribution)
  [Entrepreneurship](/Team%3ATec-Monterrey/Entrepreneurship)
* SOFTWARE

  [Overview](/Team%3ATec-Monterrey/Software)
  [Model](/Team%3ATec-Monterrey/Model)
* PARTS

  [Parts Overview](/Team%3ATec-Monterrey/Parts)[Parts Collection](/Team%3ATec-Monterrey/Parts#parts-collection)
  [Parts Improvement](/Team%3ATec-Monterrey/Parts#improved-parts)
* HUMAN

  [Human Practices](/Team%3ATec-Monterrey/Human_Practices)
  [Education and Communication](/Team%3ATec-Monterrey/Communication)
  [Inclusivity](/Team%3ATec-Monterrey/Inclusivity)
* TEAM

  [Members](/Team%3ATec-Monterrey/Team)
  [Attributions](/Team%3ATec-Monterrey/Attributions)
  [Collaborations](/Team%3ATec-Monterrey/Collaborations)
  [Sponsors](/Team%3ATec-Monterrey/Sponsors)
* [CONTACT](/Team%3ATec-Monterrey/Contact)

# NOTEBOOK

## Week 1

**August 30th to September 3rd**

* Amplified by PCR a pair of samples and tested different annealing temperatures (from 55°C to 65°C).
  + Using an annealing temperature of 60°C showed best yield.
  + Realized that not all parts were being amplified.
* Amplified all the parts we ordered in a small PCR reaction (10μL).
  + Runned electrophoresis gel to confirm amplification.
* Repeated amplification of parts shown in previous gel in a bigger reaction (50 μL).
  + Performed PCR cleanup with spin columns and quantified in Qubit fluorometer.
* Digestion of amplified parts
  + Runned electrophoresis gel to confirm amplification.
  + Performed gel DNA recovery and quantified in Qubit fluorometer.
  + Not enough DNA was recovered in order to set a cell-free reaction.
* Decided to temporarily proceed only with the pre-assembled parts.

![](https://static.igem.org/mediawiki/2021/1/1d/T--Tec-Monterrey--notebook-diftms.jpg)

**Fig.1** Electrophoresis gel of the same sample annealing at different temperatures

Next
![](https://static.igem.org/mediawiki/2021/1/1f/T--Tec-Monterrey--navigate_next_black_24dp.svg)

## Week 2

**September 6th to 10th**

* Aliquoted one of the cell free kit reagents into 40 μL reactions.
* Tested the cell-free commercial kit using a mCherry containing pET-28b plasmid in reactions of 20
  and 10 μL.
  + The reaction did not show any color, even after an overnight incubation.
* Tested again with a higher concentration of DNA.
  + Again there was not any color in the reaction.
* Tested again with a new kit in order to discard any manipulation error, this time also using one of
  our Toeholds plus Trigger as templates, as well as the mCherry containing plasmid.
  + The reaction was again unsuccessful,
    **we attribute it to bad shipping conditions.**

![](https://static.igem.org/mediawiki/2021/thumb/c/cd/T--Tec-Monterrey--notebook-cellfreebad.jpg/800px-T--Tec-Monterrey--notebook-cellfreebad.jpg)

**Fig.2** Cell-free expression reactions after an overnight incubation, negative
control is on the right.

![](https://static.igem.org/mediawiki/2021/6/60/T--Tec-Monterrey--navigate_before_black_24dp.svg)
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## Week 3

**September 13th to 17th**

* Developed a plan for testing our Toeholds in bacteria, instead of a Cell-Free system for the proof
  of concept **as we could not have access to another kit.**
  + Decided to do a cotransformation of a plasmid containing our Toehold sequence and another
    plasmid containing the corresponding Trigger sequence.
  + The plasmids to use are pSB3C5, a low copy plasmid carrying a chloramphenicol resistance gene to
    contain our Toehold sequences, and pUC19 a medium-high copy plasmid carrying an ampicillin
    resistance marker to contain our Triggers.
  + As all of our parts were flanked by M13 sequences and our pre-assembled parts did not contained
    any restriction site we could use to clone them, we ordered M13 primers with EcoRI and PstI
    restriction sites as overhangs in order to amplify the parts and clone them into their
    corresponding plasmid.

![](https://static.igem.org/mediawiki/2021/thumb/0/0c/T--Tec-Monterrey--notebook-replan.jpg/800px-T--Tec-Monterrey--notebook-replan.jpg)

**Fig. 3** Adapting experimental design

![](https://static.igem.org/mediawiki/2021/6/60/T--Tec-Monterrey--navigate_before_black_24dp.svg)
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## Week 4

**September 20th to 24th**

* Prepared DH5-α and BL21 DE3 *E.coli* competent cells for cloning and expression respectively.
  + They were stored at -80°C for a day before use.
* Tested the competent cells transforming the DNA from the Competent Cell Test Kit.
  + DH5-α cells had a high number of colonies.
  + Bl21 cells produced almost no colonies (1-3 per plate)
* As the BL21 competent cells did not grow many colonies they were tested again using a more
  concentrated DNA.
  + Using DNA from miniprep (>1μg/μL) showed a good yield.
* iGEM part from the distribution kit containing pSB3C5 plasmid with RFP insert (Distribution kit 2021
  plate 6, well 4C) as well as a pUC19 vector from NEB (Catalog #N3041) were transformed into DH5-α
  competent cells.
  + Colonies from transformation were picked and grew in LB media overnight.
* Prepared glycerol stocks from overnight culture and performed miniprep from the leftover.
  + Resulting DNA was quantified in NanoDrop.
* Performed double digestion protocol with EcoRI and PstI.
  + Runned electrophoresis gel to confirm digestion.
  + Performed gel DNA recovery and quantified in NanoDrop.
    - Very low quantity of DNA was recovered (<50ng per sample).
* Repeated digestion of minipreps using more DNA (5 μL).
  + Confirmed digestion with gel electrophoresis and recovered the digested DNA.
    - Yield was much higher (>150 ng per sample).
* Stored digested vectors until needed.

![](https://static.igem.org/mediawiki/2021/thumb/7/78/T--Tec-Monterrey--notebook-gel-digfinales.jpg/715px-T--Tec-Monterrey--notebook-gel-digfinales.jpg)

**Fig. 4** Electrophoresis gel of digestions of toeholds and triggers amplifications

![](https://static.igem.org/mediawiki/2021/6/60/T--Tec-Monterrey--navigate_before_black_24dp.svg)
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## Week 5

**September 27th to October 1st**

* Decided to try again a cell-free expression reaction using a “homemade” *E.coli* extract
  instead of the one from the kit.

+ Incubated 2X YPTG media inoculated with BL21 DE3 and induced with IPTG for an hour.
+ Concentrated the culture and sonicated the sample.
+ Plated the resulting lysate in LB agar plates in order to confirm absence of bacteria.
  - Colonies were present in plates.

* Repeated lysate production and plated it in LB Agar again.
  + **No colonies were observed this time.**
* Tested commercial kit with our *E.coli* lysate using the same mCherry PET-28b plasmid as
  template in reactions of 15 μL.
  + No color changes were observed again.
* Received M13 primers with restriction sites overhangs and started amplification of pre-assembled
  Toeholds + reporter gene as well as our triggers.
  + Runned part of the amplification in an electrophoresis gel.
  + Purified rest of the product using spin columns.
* Digested PCR products with EcoRI and PstI.
  + Runned electrophoresis gel to confirm digestion.
  + Performed gel DNA recovery and quantified in NanoDrop and good yield was observed (>250 ng).

![](https://static.igem.org/mediawiki/2021/thumb/d/d7/T--Tec-Monterrey--notebook-placalisado.jpg/307px-T--Tec-Monterrey--notebook-placalisado.jpg)

**Fig. 5** *E.coli* lysate in LB agar plate (up) and negative control (down)

![](https://static.igem.org/mediawiki/2021/6/60/T--Tec-Monterrey--navigate_before_black_24dp.svg)
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## Week 6

**October 4th to 8th**

* Ligated Toehold constructs into pSB3C5 vector and Triggers into pUC19 vector in a 10μL reaction at
  25°C for 4 hours.
* Transformed 4μL of ligation in DH5-α competent cells.
  + Blue-white screening was performed with ligations in pUC19 plasmid in order to confirm correct
    insertion by disruption of LacZα gene.
    - In ligations in pSB3C5 plasmid the original plasmid contained an RFP producing insert, so
      red colonies were avoided.
    - White colonies from transformation were picked and grew in LB media overnight.
* Prepared glycerol stocks from overnight culture and performed miniprep from the leftover.
  + Resulting DNA was quantified in NanoDrop.
* Due to high DNA concentration (>5μg/μL) from minipreps, 1:100 dilutions in Nuclease-free water were
  prepared prior to transformation.
* Transformed single plasmids and co-transformed Toeholds containing plasmids with their corresponding
  Trigger containing plasmid in BL21 DE3 competent cells.
  + 1:1 ratio of Toehold and Trigger were used.
  + Single plasmid transformations were successful but co-transformations yielded no colonies.
* Colonies from successful transformations were picked and grew in LB media overnight.
  + Prepared glycerol stocks from overnight culture.
* Repeated co-transformations using 15% less antibiotic concentration and 3X the amount of DNA.
  + Only the co-transformation of *Fusarium oxysporum* Toehold + Trigger yielded colonies.

![](https://static.igem.org/mediawiki/2021/c/cb/T--Tec-Monterrey--notebook-bluewhite.jpg)

**Fig. 6** Blue-white screening of one of our trigger ligations into pUC19 vector

![](https://static.igem.org/mediawiki/2021/6/60/T--Tec-Monterrey--navigate_before_black_24dp.svg)
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## Week 7

**October 11th to 15th**

* Colonies from successful transformation were picked and grew in LB media overnight.
  + Prepared glycerol stocks from overnight culture.
* IPTG induction was performed in culture.
  + No change in color was observed.
* Performed a colony PCR with M13 primers in order to confirm presence of both Toehold and Trigger
  sequences.
  + Runned electrophoresis gel to confirm.
    - Bands were observed in the expected Trigger size
      **but not in the Toehold expected region, suggesting its absence.**
* Single enzyme digestion (linearization) of miniprep DNA was performed with EcoRI.
  + Runned electrophoresis gel to confirm expected sizes.
    - Bands in the expected regions were observed except in one of the samples of the pSB3C5
      plasmid containing the *Fusarium oxysporum* Toehold which was observed to have the size
      of the bear backbone (this sample was the one used for transformation).
      * This suggests that the sticky ends from EcoRI and PstI digestion were somehow ligated
        resulting in the removal of the RFP CDS (thus producing a white colony) and no DNA were
        inserted.
        + This also explains how the bacteria were able to grow in antibiotic containing media
          while not carrying the desired plasmid.
* As the rest of the co-transformations produced no colonies, competent cells from successful single
  transformations were produced in order to do a serial transformation.
  + Prepared only from Toehold containing cells.
* Transformed Toehold containing competent bacteria with Trigger containing plasmids.
  + Only *Agave tequilana* and Bois Noir Toehold + Trigger transformations yielded colonies.
* Performed colony PCR to successful transformations using M13 primers.
  + Runned electrophoresis gel to confirm desired parts.
    - Bands were observed in the expected Toehold size but only a faint stain was observed in the
      Trigger expected region.
      * Decided to continue anyway.
* Colonies from successful transformations were picked and induced with IPTG.
  + No change in color was observed, even after an overnight incubation.
    - This could be due to low trigger concentration or absence as suggested by the
      electrophoresis gel.

![](https://static.igem.org/mediawiki/2021/9/91/T--Tec-Monterrey--notebook-ind-nocolor.jpg)

**Fig. 7** Pellet of induced culture of cotransformation of *Agave tequilana*'s
toehold + trigger (note the lack of expression of the RFP).

![](https://static.igem.org/mediawiki/2021/6/60/T--Tec-Monterrey--navigate_before_black_24dp.svg)
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# Team:Tec-Monterrey/Engineering

[![Main logo](https://static.igem.org/mediawiki/2021/3/39/T--Tec-Monterrey--logo-diagnosgene-blanco-stretch.svg)](/Team%3ATec-Monterrey)

* PROJECT

  [Description](/Team%3ATec-Monterrey/Description)
  [Design](/Team%3ATec-Monterrey/Design)
  [Notebook](/Team%3ATec-Monterrey/Notebook)
  [Experiments](/Team%3ATec-Monterrey/Experiments)
  [Proof of Concept](/Team%3ATec-Monterrey/Proof_Of_Concept)
  [Engineering Success](/Team%3ATec-Monterrey/Engineering)
  [Implementation](/Team%3ATec-Monterrey/Implementation)
  [Safety](/Team%3ATec-Monterrey/Safety)
  [Contribution](/Team%3ATec-Monterrey/Contribution)
  [Entrepreneurship](/Team%3ATec-Monterrey/Entrepreneurship)
* SOFTWARE

  [Overview](/Team%3ATec-Monterrey/Software)
  [Model](/Team%3ATec-Monterrey/Model)
* PARTS

  [Parts Overview](/Team%3ATec-Monterrey/Parts)[Parts Collection](/Team%3ATec-Monterrey/Parts#parts-collection)
  [Parts Improvement](/Team%3ATec-Monterrey/Parts#improved-parts)
* HUMAN

  [Human Practices](/Team%3ATec-Monterrey/Human_Practices)
  [Education and Communication](/Team%3ATec-Monterrey/Communication)
  [Inclusivity](/Team%3ATec-Monterrey/Inclusivity)
* TEAM

  [Members](/Team%3ATec-Monterrey/Team)
  [Attributions](/Team%3ATec-Monterrey/Attributions)
  [Collaborations](/Team%3ATec-Monterrey/Collaborations)
  [Sponsors](/Team%3ATec-Monterrey/Sponsors)
* [CONTACT](/Team%3ATec-Monterrey/Contact)

# ENGINEERING SUCCESS

## Software

As described in [contributions](https://2021.igem.org/Team%3ATec-Monterrey/Contribution),
during the development of our project we encountered several difficulties
in the process of designing in silico RNA toehold switches, mainly because there were several bioinformatic
tools and the process from passing information from one tool to the next became time consuming and difficult
for persons with no coding experience. Therefore we utilized the Design→Build→Test→Learn Cycle to develop an
all-in-one software tool capable of designing and analyzing both primers and toehold switches.

But once we developed the first version of our software (Toehold Switch Creator) and started testing it on different
operating systems, we found out that the overall installation process was tedious and difficult for Windows users.
Therefore we started the cycle again and we came up with the idea of developing a web application of the software so
that users could easily design their sequences without the need of installing several bioinformatic packages. And currently,
we are in the building and testing process of the web app with different cloud services.

![](https://static.igem.org/mediawiki/2021/0/03/T--Tec-Monterrey--engineering-suc.png)

## Wet Lab

Our original plan consisted of testing our toehold switches in a commercial cell-free protein expression system,
however, as noted in [notebook](https://2021.igem.org/Team%3ATec-Monterrey/Notebook) it did not work out. We needed an alternate plan so we came back to the experiment
design phase of the project and set up from scratch a completely different way to test and characterize our toeholds,
we decided to do it in bacteria. In the middle of the laboratory work phase we designed primers with needed restriction
sites overhangs, we successfully added those sites to our constructs and finally cloned them into a set of plasmids useful
to do a simultaneous transformation (cotransformation).

![]()

Our secondary plan of performing a cotransformation did not go as planned, we only got to perform single plasmid transformation
successfully so we came with the idea of growing these single plasmid containing bacteria and make them competent so we could do
a serial transformation adding the second plasmid afterwards which we finally were able to do.

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# Team:Tec-Monterrey/Experiments

[![Main logo](https://static.igem.org/mediawiki/2021/3/39/T--Tec-Monterrey--logo-diagnosgene-blanco-stretch.svg)](/Team%3ATec-Monterrey)

* PROJECT

  [Description](/Team%3ATec-Monterrey/Description)
  [Design](/Team%3ATec-Monterrey/Design)
  [Notebook](/Team%3ATec-Monterrey/Notebook)
  [Experiments](/Team%3ATec-Monterrey/Experiments)
  [Proof of Concept](/Team%3ATec-Monterrey/Proof_Of_Concept)
  [Engineering Success](/Team%3ATec-Monterrey/Engineering)
  [Implementation](/Team%3ATec-Monterrey/Implementation)
  [Safety](/Team%3ATec-Monterrey/Safety)
  [Contribution](/Team%3ATec-Monterrey/Contribution)
  [Entrepreneurship](/Team%3ATec-Monterrey/Entrepreneurship)
* SOFTWARE

  [Overview](/Team%3ATec-Monterrey/Software)
  [Model](/Team%3ATec-Monterrey/Model)
* PARTS

  [Parts Overview](/Team%3ATec-Monterrey/Parts)[Parts Collection](/Team%3ATec-Monterrey/Parts#parts-collection)
  [Parts Improvement](/Team%3ATec-Monterrey/Parts#improved-parts)
* HUMAN

  [Human Practices](/Team%3ATec-Monterrey/Human_Practices)
  [Education and Communication](/Team%3ATec-Monterrey/Communication)
  [Inclusivity](/Team%3ATec-Monterrey/Inclusivity)
* TEAM

  [Members](/Team%3ATec-Monterrey/Team)
  [Attributions](/Team%3ATec-Monterrey/Attributions)
  [Collaborations](/Team%3ATec-Monterrey/Collaborations)
  [Sponsors](/Team%3ATec-Monterrey/Sponsors)
* [CONTACT](/Team%3ATec-Monterrey/Contact)

# EXPERIMENTS

## 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

## DNA 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

**Procedure**

* 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

**Procedure**

* 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.

## Cell-Free protein synthesis

Using a positive control template to verify protein synthesis can be useful when unfamiliar with in
vitro transcription-translation protocols. To prevent nuclease contamination, wear gloves and use
nuclease-free tubes and tips. Keep all reagents on ice before and during the assembly of reactions and
avoid repeated freeze-thaw cycles of the tubes. Reactions are typically 50 μL but can be scaled down or
up, as needed. Reactions are typically assembled in nuclease-free 1.5 mL microcentrifuge tubes.
Components can be pre-assembled to create a master mix for a desired number of reactions. Use the master
mix immediately, discard any unused master mix.

**Procedure**

* Thaw all components on ice.
* Gently vortex the NEBExpress® S30 Synthesis Extract and Protein Synthesis Buffer to mix.
* Combine reagents in a 1.5 mL microcentrifuge tube on ice as follows:

  | Component | 50 µL REACTION |
  | --- | --- |
  | NEBExpress® S30 Synthesis Extract | 12 µL |
  | Protein Synthesis Buffer (2X) | 25 µL |
  | T7 RNA Polymerase | 1 µL |
  | RNase Inhibitor, Murine | 1 µL |
  | Plasmid template (>100 ng/μl) | 250 ng |
  | Nuclease-free Water | to 50 µL |
* Incubate reactions at 37°C, with vigorous shaking (250 RPM), for 2-4 hours.
* Analyze by method of choice or freeze at -20°C for later use.

## Competent cell 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

**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

**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.

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# Team:Tec-Monterrey/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)