Team:Tec-Monterrey/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.

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

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.

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

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.

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.

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

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).

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

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.

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

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.

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