Team:Edinburgh/Project/Experiment


Team Edinburgh Finding NEMO

Experiments



General lab workflow

The promoters, riboswitch, F30 scaffold and iSpinach sequences can be ordered easily from IDT. Each part is flanked by two PhytoBrick fusion sites on both sides, the whole construct can be assembled easily through ligation. The ligated construct was checked by 2% agarose gel before preforming the in vitro transcription.



Step 1: Phosphorylation and annealing

The protocol used for phosphorylation and annealing is adapted from Maryia Trubitsyna’s work (1).



  1. Spin the tubes containing the lyophilized oligonucleotides briefly (30 s, 2000 g) to ensure that the pellet is at the bottom of the tube.
  2. Add nuclease free water to obtain a final concentration of 100 μM. Mix by flicking the tube, and spin briefly (30 s, 2000 g) to ensure that all liquid is at the bottom of the tube. Place the tubes on ice.
  3. To phosphorylate oligonucleotides, combine the following in a 0.2 ml clear PCR tube:
  4. Mix by flicking the tubes, and spin briefly (30 s, 2000 g) to return all liquid to the bottom of the tube. Place the tubes in a heat-block and incubate for 30 min at 37°C.
  5. To anneal, place the tubes with the phosphorylated oligonucleotides in a heat-block at 95°C. Slowly cool down the block to room temperature from 95°C by turning off the heat-block.




Step 2: Ligation

The protocol used for ligation is adapted from Maryia Trubitsyna’s work (1).



In all our designs, ligation was required to connect four parts, promoters, F30 upstream scaffold, iSpinach aptamer and F30 downstream scaffold. Two parts were ligated at the same time in the same tube. In total, two ligation steps were performed.




  1. To ligate, combine the following in a 0.2 ml clear PCR tube:
  2. Flick tube and spin for 30 s. Incubate at 16℃ for 1 h






  1. Mix two reaction systems in equal molar into a same tube. Incubate at 16℃ for 1.5 h
  2. Then, inactivate the ligase at 65℃ for 10 mins.


Step 3: check the ligation product

Samples ran in equal mass on 2% gel, which was made by 50ml TAE or TBE buffer + 1g agarose+0.5μl SYBR safe (10000ⅹ)



T7 basic full construct (90V for 30 mins)

T7 basic full construct (90V for 30 mins)

ArsR upstream full construct (90v, 35mins)

ArsR upstream full construct (90v, 35mins)

F riboswitch construct (90v, 30mins)

F riboswitch construct (90v, 30mins)



Step 4: in vitro transcription

Because the ArsR transcription factor can be found in both E. coli K12 MG1655 and Cupriavidus metallidurans CH34 systems. Two arsenic sensing constructs were tested in both cell extracts. E. coli K12 MG1655 is one of the most common bacteria strains in the lab, which makes the procedure more accessible to the public, and Cupriavidus metallidurans CH34 was reported by Felipe to have better metal sensing ability (2).



E. coli K12 MG1655 extract was prepared using the following method. The extract for Cupriavidus metallidurans CH34 was kindly provided by Dr. Mengxi Li, that was prepared using the same method.

Cell extract preparation:





Cell culture
  1. E. coli (K12 MG1655) was grow on agar plate and incubate at 37℃ overnight
  2. One colony was picked from the plate and incubated in 300 ml M9 minimum culture at 160 rpm, 37℃ overnight
  3. Cell cultures were collected at OD600 = 2, into six 50ml falcon tubes
  4. Cells were pelleted by spinning at 4500 RPM for 20 mins at 4℃


Cell lysis
  1. Tubes of pelleted cells were placed on ice
  2. 3ml of non-ionic lysis buffer [0.1%v/v Triton X-100 (Sigma-Aldrich #T9284), 40 mM Tris base adjusted to pH 7.5 with HCl and 50 mg/mL lysozyme (Sigma-Aldrich #L6876)] was added to each pellet for cell resuspension
  3. All tubes were washed with extra 1ml non-ionic lysis buffer
  4. All the resuspended cells were transferred into a new 50 ml falcon tube.
  5. 0.5mg/ml of lysozyme (stock conc: 50mg/ml) was added into cells and incubated on ice (4℃) for 1 h to allow the reaction to fully act
  6. Centrifuge at 4℃, 10000 ⅹg for 20 mins. Collect supernatant in twenty 1ml Eppendorf tubes, labelled from 1-20.
  7. Tube 1 and 2 were used for Bradford assay to determine protein concentration.


Bradford assay :
  1. The procedure performed was exactly following the online protocol . Protein standard used was already prepared by Dr. Mengxi Li.


    2. The final protein concentration determined is ∽2.5 mg/ml for E. coli MG1655.

    The protein concentration in Cupriavidus metallidurans CH34 is ∽1.6 mg/ml.



    IVT



    In vitro transcription experiment was preformed based on conditions optimized by Dr. Mengxi Li in her cell free system. Tests for T7 basic construct was performed without the cell extract and metal, the volume was replaced by water. Both arsenic constructs were tested in MG1655 and CH34 extracts. The F riboswitch was tested in MG1655 cell extract.


    *OTRDB: stands for optimized transcription/detection buffer. 10X stock was prepared by Dr. Mengxi Li as follows: Tris base 400 mM pH 7.5 adjusted with HCl (Sigma-Aldrich #T1503); MgCl2·6H2O 60 mM (Sigma-Aldrich #M2670); DTT 100 mM (Melford #MB1015) and Spermidine 20 mM (Alfa Aesar #A19096.03) in nuclease-free water



    The fluorescence image was captured under a Safe Imager™ Blue-Light Transilluminator (Invitrogen) with an amber filter unit and a phone camera. The exact fluorescent intensity was measured by FLUOstar Omega (BMG LABTECH) in CORNING 384 LOW VOL FL. BOTTOM plate under the wavelength of 485/520nm, at 25°C with 200 rpm double orbital shaking 1s before each cycle.

    The normalized fluorescence is calculated using equation (1):

    References

    1. Trubitsyna M, Honsbein A, Jayachandran U, Elfick A, French C. PaperClip DNA Assembly: Reduce, Reuse, Recycle. Methods in Molecular Biology. 2020;:161-177.
    2. Millacura FA, Li M, Valenzuela-Ortega MA, French C. TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology. bioRxiv. 2019:826230