Notebook

Toeholds Testing – Mammalian Cell-lines

  • Lab space and instrumentation for working with mammalian cell lines were provided courtesy of Prof. Dan Peer’s laboratory (Lab website).
  • Routine handling of the mammalian cell lines is not specified in the lab notebook (cells were grown in complete DMEM and handled according to standard procedures (see ATCC guidelines).
  • Our Protocols can be found here

May 1, 2022

  • miRNA-155 and miRNA-21 expression plasmids ordered.
  • Chemically induced competent E. coli (D5H) were transformed via heat-shock with Wang and colleagues' miR-155 Toehold plasmid1, provided by courtesy of Shue Wang’s Laboratory at UNH, (Lab website) and with the two miR plasmids and were then plated in LB-ampicillin plates.

May 2, 2022

  • Colonies from plates were collected into LB+Amp.

May 3, 2022

  • Plasmids were extracted from tubes and sent for Sanger sequencing (using the T7 primer).
  • 25% glycerol stocks were made and put in -80oc.

May 9, 2022

  • HEK293 cells were seeded in a 24-well plate (2*105 cells/well), to prepare for an evaluation of Wang and colleagues’ toehold (1).

May 10, 2022

  • Cells were transfected with either miR-155 toehold alone or with miRNA-155 expression plasmid and the toehold together.
  • 100ng DNA from each plasmid was used for this experiment.

May 11, 2022

  • Cells were collected, washed with PBS, and run through FACS analysis via CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA).
  • Exhibited no GFP expression in any condition, therefore higher DNA concentrations were used for latter experiments.

May 15, 2022

  • HEK293 cells were seeded in a 24-well plate for another test of Wang and colleagues’ toehold (1).

May 16, 2022

  • Cells were transfected with either miR-155 toehold or with miRNA-155 expression plasmid and the toehold together (400 ng DNA from each plasmid).

May 17, 2022

  • Cells were collected, washed with PBS, and run through FACS analysis.
  • Exhibited no GFP expression in any condition, therefore DNA concentrations were further elevated for the next experiment.

May 23, 2022

  • HEK293 cells were seeded in a 24-well plate for the third test of Wang and colleagues’ toehold.

May 24, 2022

  • Following the first two unsuccessful runs, several combinations were tested for the third:
    • miR-155 mToehold
      1     800ng     800ng  
      2     400ng     800ng  
      3     800ng     400ng  
  • The three combinations were transfected to cells alongside a positive GFP control (using an existing GFP expression plasmid) and a toehold-only well (this time with 800 ng DNA).

May 25, 2022

  • Exhibited no GFP expression in any condition but the positive GFP control.
  • Results were first addressed as indicative of a problem in the toehold’s initial design and our first-cycle toeholds were designed according to this assumption.

May 30, 2022

  • 1st cycle sequences generated by our modeling team were ordered (Supplementary - cycles’ sequences).

June 7, 2022

  • Sequences arrived.

June 14, 2022

  • Tried to run a restriction on Wang and colleagues’ miR-155 Toehold plasmid to generate a cloning backbone:
    •   ddH2O     41 µL  
      CutSmart buffer     5 µL  
      XhoI     1 µL  
      BamHI     1 µL  
      Plasmid     2 µL  
  • Ran the gel and cut it.

June 15, 2022

  • Gel purification on the cut segment, concentration was too low.
  • Tried the restriction again with similar conditions but the band in the gel was too weak for cutting.

June 16, 2022

  • Reran the restriction in June with a different mixture (higher DNA and enzymes conc.).
    •   ddH2O     19.5 µL  
      CutSmart buffer     2.5 µL  
      XhoI     1 µL  
      BamHI     1 µL  
      Plasmid     2 µL  
  • The gel showed 2 weak bands, decided to cut the lower one as the size indicated this is the correct one.
  • After Purifying the gel slices, we were only able to achieve a DNA conc. Of 13.8ng/ul.

June 19, 2022

  • Ran Gibson using the gel output plasmid and the open GFP according to the following ratios:
    •   DDW (ul)     0  
      Vector (ul)     3.6 µL  
      Open GFP fragment #1 (ul)     0.5  
      Mid-End GFP #2 (ul)     0.9  
      Gibson Assembly Mix (ul)     5  
  • The reaction was run at 50C for 1 hour as per protocol and left overnight in 4C.

June 20, 2022

  • Transformations with Gibson results. were run – cells underwent transformation protocol using either 5ul of the Gibson or 50ng of the cut plasmid (3.6ul cut plasmid + 1.4ul DDW) as control.

June 21, 2022

  • Neither plate showed any colonies, leading us to think that something was wrong with the cut segment.

June 22, 2022

  • Another attempt to run the restriction using 3ug plasmid. A cut band was observed in the gel, but when purified the Nanodrop measurements showed a yield too low to proceed.

July 12, 2022

  • We attempted to switch to the buffer when performing the restrictions. Reran the restriction using the FastDigest buffer instead of the Cutsmart.

July 13, 2022

  • We ran the gel and saw the proper band. However, purification gave a low concentration.
  • After the revision of gel pictures, we observed a big band seen in several previous gels indicating that something might be wrong with the plasmids used.

July 20, 2022

  • We decided to try amplifying the required segment using PCR instead and primers were ordered (LINK).

July 27, 2022

  • Primers arrived.
  • PCR was attempted using the Kapa HiFi HotStart Readymix PCR kit (0.75 of each primer, 12.5ul of PCR mix, 10ul DDW and 1ul Plasmid).
    •   ddH2O     10 µL  
      PCR mix     2.5 µL  
      Plasmid     1 µL  
      Primer (each)     0.75 µL  
  • The PCR was run using the following program:
    •   95℃     3 min.     X1  
      98℃     20 sec.     X35  
      66℃     15 sec     X35  
      72℃     3 min.     X35  
      72℃     1 min.     X1  

July 30, 2022

  • PCR results were tested on Gel, which showed a smear that existed on both the no-template control and the reaction:

Aug 1, 2022

  • PCR was repeated with 68oc instead of 66oc annealing. The results in the gel again showed only a smear.
  • Our struggle to complete the cloning process led us to consider a problem with the plasmid’s integrity in the glycerol stock. Therefore, PCR was tested on both 66℃ and 68℃ using an old plasmid that was harvested from the original starter from which the glycerol was made (13ng).
  • This time, the gel for the old plasmid showed a clear band in the appropriate position’ and the ‘uncut’ plasmid.
  • The bands were purified and gave 38.7 ng/ul. The resulting DNA fragment was used in a Gibson reaction according to the following table:
    •   DDW (ul)     2.6  
      Vector (ul)     1.4  
      Open Toehold fragment (ul)     0.5 sec  
      Mid-End Fragment #2 (ul)     0.5  
      Gibson Assembly Mix (ul)     5  
  • Half of the Gibson reaction was then passed through a restriction reaction with XhoI as that is supposed to get rid of any theoretical original plasmid. Incubated for 1 hr.
    •   ddH2O     3 µL  
      FastDigest buffer     1 µL  
      XhoI     1 µL  
      Gibson output     5 µL  
  • The transformation was performed on 3 Amp-containing plates: 1 with 5ul of the restriction results, 1 with the remaining 5ul of uncut Gibson and 1 with 400ng of the original old plasmid (both as a positive control and since a new glycerol stock is needed).

Aug 2, 2022

  • Colonies were observed only on the mir155-Toehold plate, indicating that something went wrong with the Gibson reaction. From this plate, 2 colonies were picked for starters (3ml LB + 3ul amp).

Aug 3, 2022

  • we decided to repeat the Gibson reaction with increased vector-to-insert ratios:
    •   DDW (ul)     2.6  
      Vector (ul)     1.4  
      Open Toehold fragment (ul)     0.5 sec  
      Mid-End Fragment #2 (ul)     0.5  
      Gibson Assembly Mix (ul)     5  
  • Half of the Gibson reaction was then passed through a restriction reaction with XhoI as that is supposed to get rid of any theoretical original plasmid. Incubated for 1 hr.
    •   1-to-3     1-to-4  
      ddW (µL;L)     2.15     1.7  
      Vector (µL;L)     1.4     1.4  
      Insert#1 (µL;L)     0.75     1  
      Insert#2 (µL;L)     0.7     0.9  
      Gibson Assembly Mix (µL;L)     5     5  
  • Transformation was performed with both plasmids.

Aug 4, 2022

  • The transformation plates showed many colonies from each reaction.

Aug 10, 2022

  • 2 colonies from each plate were extracted to LB starters.

Aug 11, 2022

  • Half of the starters were harvested and used to make 25% Glycerol stocks, stored in -80℃.
  • 1 plasmid from each plate was sent to sequencing.

Aug 12, 2022

  • both showed the original plasmid.
  • Several other colonies were sent to sequencing using the F primer to see what comes out at the beginning of the plasmid.

Aug 14, 2022

  • PCR was rerun using less template (6.5ng vs 13ng last time) and run through gel.

Aug 15, 2022

  • Plasmids were purified on and sent for sequencing.

Aug 16, 2022

  • Only one starter gave the right plasmid (from the open GFP sequence) – therefore we gave it a short recovery session in the shaker before proceeding to make the glycerol.

Aug 17, 2022

  • Gibson reactions were assembled for the other 5 plasmids according to the following table:
    •   1.1     1.2     1.3     1.4     1.5  
      DDW (µL;L)     1.9     2.1     2     2.1     1.9  
      Vector (35.7ng/µL;L) (µL;L)     1.4     1.4     1.4     1.4     1.4  
      Insert#1 (µL;L)     0.8     0.6     0.7     0.6     0.8  
      Insert#2 (µL;L)     0.9     0.9     0.9     0.9     0.9  
      Gibson Assembly Mix (µL;L)     5     5     5     5     5  
  • A transfection experiment was run using the newly assembled open-toehold GFP alongside wells which included either the original miR155-Toehold plasmid or both the Toehold and trigger plasmids – it was decided to test the results by FACS after 24 hours.

Aug 18, 2022

  • The results showed that the open GFP works, but similar results were observed in the miR155-Toehold with and without the trigger, so it is possible that the vial used was contaminated by the open plasmid.
  • 5µL;L of each Gibson reaction were added to a restriction reaction:
    •   ddH2O     3 µL  
      CutSmart buffer     1 µL  
      XhoI     1 µL  
      Gibson output     5 µL  
  • 1 hour at 37℃ followed by 10 minutes of inactivation at 65℃, reaction tubes were then put at -20℃.

Aug 21, 2022

  • All 5 restriction results transformed into bacteria.

Aug 22, 2022

  • All plates were empty of colonies.

Aug 23, 2022

  • Another Gibson reaction was performed – since there wasn't enough PCR product for all 5 reactions, only plasmids 1-4 were made according to the following table:
    •   1.1     1.2     1.3     1.4  
      DDW (µL;L)     0     0     0     0  
      Vector (18.1ng/µL;L) (µL;L)     2.8     2.8     2.8     2.8  
      Insert#1 (µL;L)     0.8     0.7     0.7     0.6  
      Insert#2 (µL;L)     1.9     1.9     1.9     1.9  
      Gibson Assembly Mix (µL;L)     5     5     5     5  
  • Another transformation was attempted with the Gibson results.
  • HEK-293 and mouse HEPA cells were seeded in a 24-well plate.

Aug 24, 2022

  • The plates showed many colonies.
  • As an initial screen, 2 colonies were picked from each into an LB starter.
  • Wang and colleagues miR-155-toehold-GFP (1) or a constitutive GFP plasmid were transfected to the cells.

Aug 25, 2022

  • Starters were harvested for plasmids. From each plasmid, 500ng were taken into a restriction reaction.
    •   DNA     500 µgr  
      BAmHI     1 µL  
      XhoI     1 µL  
      CutSmart     2 µL  
      ddH2O     Complete to 20 µL total  

Aug 25, 2022

  • Results were run through gel and compared to uncut plasmid extracted from the same colony.
  • Successfully cloned plasmids should be cut only once. Two colonies from sequence 3 and one from sequence four seemed to be cut only once.
  • HEK-293 and HEPA cells were collected, washed with PBS, and run through FACS analysis.

Aug 28, 2022

  • The last remaining Gibson reaction (for seq. 5) was run, according to the following table:
    •   DDW (µL)     0  
      Vector (µL)     3.1  
      Open Toehold fragment (µL)     0.8  
      Mid-End Fragment #2 (µL)     1.9  
      Gibson Assembly Mix (µL)     5  

Aug 29, 2022

  • The Gibson mix of seq. 5 was used for transformation.

Aug 30, 2022

  • No colonies were observed for the seq. 5 transformation.
  • The 3 seemingly correct plasmids were sent for sequencing on Aug 30th.

Aug 31, 2022

  • Sequencing results showed that only one of the seq 3 plasmids was correct.
  • 3 new colonies from the plates with seq 1,2, and 4 were collected into starters.
  • The starter containing the correct seq 3 was given the night to recover.

Sep 1, 2022

  • Glycerol stock was made from the seq 3 starter.
  • The rest of the starter was harvested for DNA extraction.
  • Plasmids with seq 1,2 and 4 were extracted from starters and tested by restriction with XhoI only, and the gel indicated that 2 sequences out of every triplicate might be correct.

Sep 4, 2022

  • The plasmids that seemed correct in the gel were sent for sequencing.
  • Gibson and transformation for plasmid 5 were repeated.

Sep 5, 2022

  • Sequences were received and showed that there was at least 1 correct plasmid from each set.
  • The plate for plasmid 5 showed many colonies.
  • HEK293T plate was made as per protocol for testing all 4 sequences alongside the original one.
  • All sequences were transfected to cells with or without the miR-155 trigger.

Sep 6, 2022

  • The correct plasmids from the last prep were taken into a glycerol stock following an overnight recovery.
  • The fluorescence of cells transfected with seq 1-4 and the original toehold was tested through FACS (see Results/mammalian toehold testing).

Sep 7, 2022

  • 4 more colonies from the Plasmid 5 plate were picked.

Sep 11, 2022

  • Seq 5 starters were harvested, leaving half of each.

Sep 12, 2022

  • To test whether the GFP reduction arises specifically from miR interference, an experiment was performed in which several plasmid combinations were tested, including the open GFP plasmid and the miR, and the original plasmid alongside a Luciferase expression plasmid. All sequences were transfected to HEKs according to protocol.

Sep 13, 2022

  • Transfection results were read using FACS (see Results/mammalian toehold testing).
  • Seq 5 plasmids were tested through restriction, with the gel showing a single possibly correct plasmid.

Sep 14, 2022

  • The putatively correct seq-5 plasmid was sent for sequencing.

Sep 19, 2022

  • A second transfection experiment was performed for the 4 sequences and the original toehold.

Sep 20, 2022

  • Transfection results were read using FACS (see Results/mammalian toehold testing).

Sep 27, 2022

  • A third transfection experiment was performed for the 4 sequences, the original toehold, and the open GFP toehold. This time, cells were either transfected with the sequence alone, the sequence and its miR-155 trigger, or with the sequence and miR-21, which is not supposed to bind to the sequences.

Sep 28, 2022

  • Transfection results were read using FACS (see Results/mammalian toehold testing).

References:

  1. Wang, S., Emery, N. J., & Liu, A. P. (2019). A novel synthetic toehold switch for microRNA detection in mammalian cells. ACS synthetic biology, 8(5), 1079-1088.

Toeholds Testing – Yeast

  • Instrumentation for working with Yeast was provided courtesy of Prof. Martin Kupiec’s laboratory (Lab website).
  • Our Protocols can be found here

Sep 1, 2022

  • Sequences were ordered (Supplementary - cycles’ sequences).

Sep 14, 2022

  • Yeast’s backbone plasmid (pRS425) was restricted using BamH1 and Hind3 restriction enzymes.

Sep 15, 2022

  • Sequences 1-5 for the yeast experiment arrived.
  • Yeasts were transformed with the sequences obtained and with cut backbone plasmids and seeded in SD-Leu plates.

Sep 18, 2022

  • No colonies were observed on the yeast plates.
  • Existing mCherry-expressing yeasts (courtesy of Kupiec lab, see above) were extracted to a YPD starter.

Sep 19, 2022

  • The remaining sequences arrived.

Sep , 2022

Sep 22, 2022

  • Another transformation attempt was done using pRS425 backbone plasmids from a different batch.
  • Yeasts were transformed with a different mCherry inserted into pRS426 plasmids.

Sep 23, 2022

  • Many colonies were detected on plates from the resulting transformed cells.

Oct 2, 2022

  • Transformed colonies were moved to YPD to grow overnight.

Oct 3, 2022

  • Yeast starters were washed in and transferred into 96-well plates
  • Fluorescence and OD were read in Synergy H1 Microplate reader (Agilent BioTek, CA, USA).
  • mCherry expression in the relevant populations was validated before GFP measured and compared between the different conditions (see Results)

Parts’ Optimization

  • Our Protocols can be found here

      • December 2021 – February 2022:

      • BBa_K079050 part’s sequences (expressing GFP after exposure to UV) were optimized with the ESO program (1) and ordered.
      • Bacteria were transformed with a plasmid (pSB1C3) containing the non-optimized BBa_K079050.
      • Chi.Bio (2), a robotic platform for automation of biological experiments which we wished to use both as a chemostat and a spectrophotometer, was chosen to be applied for automated evolution experiments. We attempted to measure the time it takes for the transformed bacteria to lose the fluorescent signal generated by the part when the Chi.Bio controls for constant OD to allow logistic growth and measures fluorescence.
      • Due to recurrent contaminations of samples in the ChiBio device, we have decided to proceed with a manually-administered evolution experiment.

      March – April 2022:

      • Bacteria transformed with the non-optimized part were grown in selective media (LB + Chloramphenicol) and examined for fluorescence level in a plate-reader in 24-hour intervals (See Results/Parts’ optimization).
      • DNA damage initiates the parts’ fluorescent activity when bacteria are applied with methyl methanesulfonate (MMS).

      May – June 2022:

      • Optimized sequences were cloned into pSB1C3 plasmids using restriction enzymes.
      • After several failed cloning attempts, we revised our cloning procedure and noticed that the restriction sites in the optimized sequences were too short for the enzymes chosen (EcoRI and PstI), therefore we tried to use other restriction enzymes (XbaI and SpeI), for which restriction was successful.
      • A manual evolution experiment was done as described above, with fluorescent signals measured daily (see Results/Parts’ optimization).

      References:

      1. Menuhin-Gruman, I., Arbel, M., Amitay, N., Sionov, K., Naki, D., Katzir, I., ... & Tuller, T. (2021). Evolutionary Stability Optimizer (ESO): A Novel Approach to Identify and Avoid Mutational Hotspots in DNA Sequences While Maintaining High Expression Levels. ACS Synthetic Biology, 11(3), 1142-1151.
      2. Steel, H., Habgood, R., Kelly, C. L., & Papachristodoulou, A. (2020). In situ characterisation and manipulation of biological systems with Chi. Bio. PLoS biology, 18(7), e3000794.
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