For building up DOPL LOCK, we designed and set up necessary experiments to demonstrate the proof-of-concept. In this page, you can find an overview of the experiment design and a summary of the experiments we performed.

Experimental design

Because the toxin:antitoxin ratio to maintain the balance state was hard to find in previous literature, we used a bottom-up approach to build up the DOPL LOCK system. We divided the experiments into three stages. The first stage of the experiments was focusing on the characterization of each part in the DOPL LOCK system. For the characterization of the origin of replication (Oris), we co-transformed Escherichia coli (E. coli) TOP10 with two plasmids containing different fluorescent protein expression cassettes. Based on the fluorescent intensity of the strains, the best compatible pair of Oris was selected. For the characterization of toxins, we cloned inducible promoters in front of the toxin genes and performed a toxicity assay by measuring the bacterial growth after expression of the toxins was induced. For the promoters, we ligated promoters with mCherry to measure their transcriptional activity. For the second stage, we aim to create a single toxin-antitoxin system (TA system) based on the characterization from the first stage. The single TA system contains constructs of inducible promoter::toxin and constitutive promoter::antitoxin on a single plasmid. It will be used to find the balanced expression level. In the final stage, two verified single TA systems can be assembled and the final version of DOPL LOCK can be achieved.

 Figure 1 overview of DOPL LOCK system Figure 1: overview of DOPL LOCK system

Experiment 1: sfGFP-RFP swap

We swapped the sfGFP reporter cassette in the original JUMP plasmids (comprised of constitutive promoter BBa_J23100 and sfGFP gene) to a RFP reporter cassette (BBa_J04450). This experiment created a second collection of JUMP plasmids with different reporters, which enabled us to evaluate the behavior of the co-transformed plasmids. The gene swap was done based on restriction enzyme cloning. JUMP plasmids and pSB1A3-RFP were both restricted by PstI and EcoRI, providing us with the JUMP backbone and an RFP construct. Then, the restriction products were mixed with a backbones/constructs molar ratio of 1:3. The DNA ligase and the buffer were added to the mixture for the reaction. Finally the reactions were stopped by heat shock and the products were ready for the transformations.

Experiment 2: (Co-)transformation of sfGFP and RFP plasmids

Figure 2 Selection of Oris

Figure 2: Selection of Oris

Table 1: The plasmids and their constructs used in the co-transformation

pJUMP26 Kanamycin p15A sfGFP B Medium/Low copy number
pJUMP27 Kanamycin pSC101 sfGFP C Low copy number
pJUMP28 Kanamycin pUC sfGFP A High copy number
pJUMP29 Kanamycin pBR322/ROB sfGFP A Medium/High Copy number
pJUMP46 Spectinomycin p15A RFP B Medium/Low copy number
pJUMP47 Spectinomycin pSC101 RFP C Low copy number
pJUMP48 Spectinomycin pUC RFP A High copy number
pJUMP49 Spectinomycin pBR322/ROB RFP A Medium/High Copy number

Both single- and co-transformations of JUMP plasmids were performed during our labtime. Detailed information of plasmids used in this experiment is shown in Table 1. Co-transformations were performed to test the compatibility of the different Oris on the different JUMP plasmids and therefore the stably maintenance of the plasmid (Figure 2). The sfGFP and RFP containing JUMP plasmids were co-transformed in the E.coli TOP10 strain. Co-transformations were performed by heat-shocking the sfGFP and RFP plasmids into TOP10 competent cells according to the co-transformation protocol. Next, the transformants are grown on sterile LB agar medium containing spectinomycin and kanamycin as a selection method. After successful transformation in E.coli, fluorescent markers were expressed and measured by plate reader assay. As a result, it was shown that the Oris p15A and pBR322/ROB were most suitable to express similar levels of protein (Results page).

Single-transformations were often used as a positive control during plate reader assays, containing only a sfGFP or RFP plasmid. TOP10 strain containing no plasmids was used as negative control. The same protocol was used for the co-transformation, but in this case, only one plasmid was added to the competent TOP10 cells.

Experiment 3: Biobrick ligation

Standardized parts and coding sequences needed for the DOPL LOCK system, including toxins, antitoxins, promoters, GFP, and mCherry were obtained as gBlocks from our sponsoring company Integrated DNA Technologies (IDT). The toxin gBlocks are double stranded DNA fragments that were cloned into the iGEM plasmids pSB1A3 and pSB1C3 by restriction enzyme cloning. The ligation was performed according to the Ligation of gBlock DNA into plasmids protocol by first diluting and digesting the gBlock DNA with EcoRI and PstI. Then, the digested gBlocks were ligated into the linearized pSB1A3 or pSB1C3 plasmids by EcoRI and PstI. We transformed these plasmids into DH5α strain to multiply their amounts. Ligating the gBlocks into both plasmids enables us to assemble composite parts that require the following experiments rapidly.

Experiment 4: 3A assembly

3A assembly was intensively used in our project to create constructs for the characterization of different parts. The assembly was performed according to the protocol in iGEM registry. The plasmid products of 3A assembly were transformed into E. coli and grown on plates with kanamycin or spectinomycin. Only the strain containing the correct construct could survive. They were transferred to liquid LB medium to be prepared for the measurement of the optical density or the fluorescent intensity.

We cloned a variety of promoters (the Anderson collection of constitutive promoters and inducible promoters) in front of the fluorescent protein/toxin/antitoxin genes. For the promoter characterization, we assembled plasmids of pJUMP49-pBAD::mCherry and pJUMP49-p2547::mCherry. To evaluate the TA system, we assembled pJUMP26-pBAD::ccdB, pJUMP26-pBAD::HOK, pJUMP26-pBAD::RelE and pJUMP26-pBAD::MazF.

For more information about these constructs, see Parts.

Experiment 5: Plate reader fluorescence assay

Figure 3 Selection of the promoters Figure 3: Selection of the promoters

We calibrated the inducible promoter pBAD (BBa_I0500) with the constitutive promoter BBa_J23100 (referred to as p2547 in the following) using plate reader measurements (TECAN spark) (Figure 3). By doing this, we can relate these two kinds of promoters and use them to regulate toxin-antitoxin gene expressions. The plasmids used in the calibration were pJUMP49-pBAD::mCherry, pJUMP49-p2547::mCherry, and pJUMP49-pBAD (see Parts page). These plasmids were created by 3A assembly and transformed to the E. coli TOP10 strain. A 96-well plate with a gradient of L-arabinose was prepared and strains containing correct constructs were added to the plates in triplicates (more details in Measurement page). After that, the 96-well plates were put in the plate reader. The temperature was maintained at 37 °C in the plate reader and the plate shaken at 120 rpm. Every 12 min the fluorescence and the optical density of each plate were measured for 10 h.

The plate reader was also used to evaluate the compatibility of Oris when co-transformed. The co-transformed cells were cultured overnight in LB-medium and pippeted to a 96-well plate in triplicates accordingly. Each overnight culture was continually diluted 5 times to another three wells. Then, the plate was put in the plate reader for a one-time measurement of fluorescence and optical density. The temperature and the shaking speed were the same as previously mentioned.

Experiment 6: Toxicity assay in liquid medium

For the evaluation of the toxicity of toxins, we first ligated the inducible promoter pBAD with each toxin in the pJUMP26 backbone (see Parts page). In this way, the toxin gene expression was regulated by the L-arabinose concentration in the environment. After successful transformation of the desired constructs in E. coli TOP10, the strains were inoculated in LB-medium overnight. Then, they were diluted 5 times and pipetted to a 96-well plate in triplicates. Different amounts of L-arabinose were added accordingly to each well. Ampicillin was added to some wells to achieve full cell death as positive controls. Strains containing pBAD-mCherry were used as a negative control to exclude the inhibitory effect of overexpression. The optical density of these cells was measured by plate readers continuously for 10 h to evaluate their viability after toxin expression was induced. The temperature was maintained at 37 °C in the plate reader and the plate shaken at the speed of 120 rpm. Every 12 min the fluorescence and the optical density of each plate were measured.

We also applied minimal medium instead of LB-medium for the toxicity assay. The procedures were similar as described above, except for using M9 minimal medium to prepare the 96-well plate.

Experiment 7: Toxicity assay by LB-agar plate

By plating cells with and without toxin expression, we can obtain different cell viability when toxin expression is induced. To do this, the agar plates with different amounts of L-arabinose as supplements were first prepared. Then, the strains containing different pBAD::toxin constructs were diluted until optical density reached 0.1. Then 5 μL of the bacterial solution were pipetted at the same position of the plates. The plates were put in the incubator overnight at 37 °C. The diameters of the colonies indicated cell viability with or without toxin induction.

Experiment 8: PCR-based cloning

As the promoters we used were less than 100 bp, we noticed difficulty trying to clone the promoters into plasmids. Therefore, primers were developed to ligate the p162 promoter in front of the ccdB, ccdA, RelE and RelB by PCR-based cloning (the list of the primers are shown in Table 2). For the PCR reaction a PCR gradient was performed according to PCR-based cloning protocol. To verify if the promoter was successfully ligated in front of the toxins, gel electrophoresis was performed. We succeeded in creating these constructs via PCR cloning (see Results page). Thereafter, the obtained constructs were used further trying to create the DOPL LOCK system. Although this was not successful due to time constraints, this can still be researched further in the future.

Table 2: The primers used in PCR-based cloning


Experiment 9: Competent cells preparation

Two different E.coli strains were used during our lab time, including DH5α and TOP10. DH5α was first used to test the overexpression of toxins, although the DH5α strain metabolized the arabinose. As a result, the promoters were not induced enough to cause bacterial death. The TOP10 bacterial strain was then used for the plate reader assay to induce the toxins. The protocol E.coli competent cells TSS method was used to make competent cells of both E. coli strains. First, the cells were inoculated in liquid LB medium until the culture reached an OD600 between 0.3-0.4.

After centrifugation 5 mL of cold TSS medium was added to the cells and cooled down in liquid nitrogen. Ultimately, the competent cells were stored at -80 °C and thawed before use.

  • Ligation of gBlock DNA into plasmids

    gBlock DNA of toxin, antitoxin, and promoters was obtained from IDT.


    • pSB1A3 or pSB1C3 linearized with EcoRI-HF and PstI
    • gBlock DNA, IDT
    • EcoRI-HF
    • PstI
    • T4-Ligase
    • T4-ligase buffer


    • Incubator at 37°C
    • Vortex
    • Centrifuge

    Resuspension of gBlock DNA

    Centrifuge the tube for 5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube.

    Add 20 ul AE-buffer to the IDT DNA.

    Briefly vortex and centrifuge

    Store in -20

    Digestion of gBlock DNA

    Table 1: Master mix schedule

    Components 1 reaction (µL) Master mix for X reactions (µL)
    gBlock DNA (10 ng/µL) 7 N/A
    EcoRI-HF 1 (X + 1) * 1
    PstI 1 (X + 1) * 1
    10x Cutsmart buffer 1 (X + 1) * 1
    MiliQ 0  
    Total volume 10 (X + 1) * 10

    Transfer 10µL of the gblock DNA from the Stock tube to a fresh 1.5mL tube.

    Add 20µL of the master mix to the gBlock DNA

    Vortex briefly and spin down at 3000 RPM for 1 second

    Incubate in 37°C for 1-2 hours

    Heat inactivate at 80 °C for 20 minutes.

    Use immediately or store in -20°C

    Ligation of Biobrick in Plasmid

    Mix the following components in a 1.5 mL tube, for ratios between insert and vector, check table 2, calculated via:!/ligation, a ratio of 1:5 was used.

    Incubate at RT for 2h or overnight.

    Transform in E. coli and pick the white colonies.

    Table 2: Ligation mixture

    Components 1 reaction (µL)  
    Linearized plasmid (20ng/µL) 2 30
    Digested gBlock DNA (3.33 ng/µL) 10  
    T4-ligase 1 15
    10x T4-ligase buffer 2 30
    MiliQ 4 60
    Total volume 20  

    Table 3: Overview of ligation in Biobrick plasmids

    Vector Insert        
    Name Size (KB) Amount (ng/reaction) Name Size (KB) Amount (ng/reaction)
    pSB1C3 2170 20 p1 125 5.8
    pSB1C3 2170 20 p21 125 5.8
    pSB1C3 2170 20 p162 125 5.8
    pSB1C3 2170 20 p387 125 5.8
    pSB1C3 2170 20 p623 125 5.8
    pSB1C3 2170 20 p908 125 5.8
    pSB1C3 2170 20 p1303 125 5.8
    pSB1C3 2170 20 p1487 125 5.8
    pSB1C3 2170 20 p1831 125 5.8
    pSB1C3 2170 20 p2547 125 5.8
    pSB1A3 2155 20 p_pBAD_CcdB_T_S 1700 78.9
    pSB1A3 2155 20 p_pLacI_CcdA_T_S 1673 77.6
    pSB1A3 2155 20 CcdB T 482 22.4
    pSB1A3 2155 20 CcdA AT 395 18.3
    pSB1A3 2155 20 MazF T 512 23.8
    pSB1A3 2155 20 MazE AT 425 19.7
    pSB1A3 2155 20 RelE T 470 21.8
    pSB1A3 2155 20 RelB AT 416 19.3
    pSB1A3 2155 20 HOK T 603 28.0
    pSB1A3 2155 20 SOK AT 389 18.1
    pSB1A3 2155 20 pBAD 1263 58.6
    pSB1A3 2155 20 pLacI 1323 61.4
    pSB1A3 2155 20 pTetOn 581 27.0
    pSB1A3 2155 20 LacI RFP cassette 1122 52.1
    pSB1A3 2155 20 GFP 896 41.6
    pSB1A3 2155 20 mCherry 890 41.3
    pSB1A3 2155 20 lambda t0 terminator 148 6.9
  • OD to CFU calibration

    The aim is to relate optical density measured by spectrophotometer or plate reader to colony counting units. So we can estimate the cell count directly from the OD600 value.

    OD test

    E. coli
    DH5α - Petri dishes - Eppendorf 2mL - Eppendorf 1mL - Falcon tube 15mL

    1. Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of your plate reader.
    2. Dilute your overnight culture to OD600 gradient in 1mL of LB + antibiotics media. Do this in triplicate for each culture.
    3. Use (C1)(V1) = (C2)(V2) to calculate your dilutions (C1 is your starting OD600; C2 is your target OD600 (= 0.1); V1 is the unknown volume in μL; V2 is the final volume (= 1000 μL)
    4. Dilute the OD600 to a certain value (Shown in the 96-well plate).
    5. Recommended plate setup is below. Each well should have 200 μL. Put the plate in the plate reader. And test their OD600 via spectrophotometer immediately.

    Table 4: Layout well

    OD0.05A OD0.05B OD0.05C OD0.1A OD0.1B OD0.1C            
    OD0.2A OD0.2B OD0.2C OD0.4A OD0.4B OD0.4C            
    OD0.8A OD0.8B OD0.8C                  
    Blank control A Blank control B Blank control C                  
    1. Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 3. You should have 18 total Starting Samples. For OD=0.05, dilute 1:20, 1:20, 1:10, 1:10, 1:10; For OD=0.1, dilute 1:20, 1:20, 1:20, 1:10, 1:10; For OD=0.2, dilute 1:40, 1:20, 1:20, 1:10, 1:10; For OD=0.4, dilute 1:40, 1:40, 1:20, 1:10, 1:10; For OD=0.8, dilute 1:8, 1:20, 1:20, 1:10, 1:10, 1:10
    2. You will need 3 Liters of LB Agar (45 petri dishes in total)
    3. Label each tube according to the figure above (Dilution 1, etc.) for each Starting Sample
    4. Split the LB plate in three parts and spead 33.3 μL of the sample on each part of the plate.
    5. Incubate at 37 °C overnight and count colonies after 18-20 hours of growth
    6. Count the colonies on each plate with fewer than 300 colonies
    7. Next, multiply the colony count by the Final Dilution Factor on each plate
  • Inducible promoters

    Experiments for the model and to test the amount of protein produced by the inducible promoters.

    Testing the inducible promoters

    1. Spectrophotometer
    2. Plate-reader
    3. Cells with plasmids
      1. GFP + LacI promoter
      2. GFP + pBAD promoter
      3. GFP + TetON promoter
      4. mCherry + LacI promoter
      5. mCherry + pBAD promoter
      6. mCherry + TetON promoter
    4. Inducer
    5. Media
    6. Cell counter


    Make a calibration curve between OD600 (0 - 1.5) and number of cells by measuring the OD & cell count of different values.

    Make a reference for the fluorescence and amount of GFP protein.


    Transform the cells with the plasmids

    Dilute the cells to about 0.1 and add them in the 96-well plate

    Grow the cells up to OD600 = 0.5

    Add certain concentration inducer (according to the table below)

    Measuring Fluorescence

    Measure the fluorescence and OD600 every 10 minutes during 5 hours with the plate reader

    Table 5: Well layout

    C1 C1 C1 B1 B1 B1 L1 L1 L1 T1 T1 T1
    C2 C2 C2 B2 B2 B2 L2 L2 L2 T2 T2 T2
    C3 C3 C3 B3 B3 B3 L3 L3 L3 T3 T3 T3
    C4 C4 C4 B4 B4 B4 L4 L4 L4 T4 T4 T4
    C5 C5 C5 B5 B5 B5 L5 L5 L5 T5 T5 T5
    C6 C6 C6 B6 B6 B6 L6 L6 L6 T6 T6 T6
    C7 C8 C9 B7 B7 B7 L7 L7 L7 T7 T7 T7
          B8 B8 B8 L8 L8 L8 T8 T8 T8

    Table 6: Overview

    C1 Media with antibiotics
    C2 Media with antibiotics with E. coli without plasmid
    C3 Media with antibiotics with E. coli with pBAD
    C4 Media with antibiotics with E. coli with LacI
    C5 Media with antibiotics with E. coli with TetON
    C6 Media with antibiotics with E. coli with plasmid
    C7 E. coli with pBAD - 0.0 mM
    C8 E. coli with lacI - 0.0 mM
    C9 E. coli with TetON - 0.0 Mm
    B1 E. coli with pBAD - 0.01 mM
    B2 E. coli with pBAD - 0.02 mM
    B3 E. coli with pBAD - 0.05 mM
    B4 E. coli with pBAD - 0.1 mM
    B5 E. coli with pBAD - 0.2 mM
    B6 E. coli with pBAD - 0.5 mM
    B7 E. coli with pBAD - 0.8 mM
    B8 E. coli with pBAD - 1 mM
    L1 E. coli with lacI - 0.01 mM
    L2 E. coli with lacI - 0.02 mM
    L3 E. coli with lacI - 0.05 mM
    L4 E. coli with lacI - 0.1 mM
    L5 E. coli with lacI - 0.2 mM
    L6 E. coli with lacI - 0.5 mM
    L7 E. coli with lacI - 0.8 mM
    L8 E. coli with lacI - 1 mM
    T1 E. coli with TetON - 0.01 mM
    T2 E. coli with TetON - 0.02 mM
    T3 E. coli with TetON - 0.05 mM
    T4 E. coli with TetON - 0.1 mM
    T5 E. coli with TetON - 0.2 mM
    T6 E. coli with TetON - 0.5 mM
    T7 E. coli with TetON - 0.8 mM
    T8 E. coli with TetON - 1 mM
  • FACS preparation

    Prepare FACS sample

    1. FACS tubes + filter/strainer (40 um)
    2. Cell culture
    3. PBS
      1. NaCl: 137 mM
      2. KCl: 2.7 mM
      3. Na2HPO4: 10 mM
      4. KH2PO4: 1.8 mM
      5. All in MiliQ water

    Spin-off 1 mL of cell suspension (OD between 0.1-0.5 but higher is also okay)

    Take away supernatant

    Resuspend in 1 mL PBS

    Drop onto 40 um filter (presoaked in PBS)

    200 uL of the filtered liquid can be put through the FACS machine

    Running the FACS

    Startup the machine (before sample prep)

    When the machine is warmed up, select protocol

    Screenshot settings

    Run blank to find background

    Put 200 uL sample in cell sorter tubes

  • 3A Assembly

    Assembly promoter and insert gene into new plasmid backbone


    calculate the amount of plasmid and MilliQ needed for each digestion using the NEB calculator for your desired molar ratios. The molar ratio needed depends on the length of the inserts and backbone.


    Find corresponding restriction enzymes for each plasmid.

    Plasmid backbone: EcoRI, PstI

    Promoter: EcoRI, SpeI

    Insert gene: Xbal, PstI

    The ligation solution should contain the following parts (uL):

    Plasmid concentration need to be measured in advanced

    Plasmid should be added at last

    Enzymes and smartbuffer should always be put on ice and stored immediately after it once has been used

    Table 7: Digestion mixture

    Enzyme 1 0.5   Plasmid ID Plasmid concentration Plasmid water
    Enzyme 2 0.5     0 x x
    buffer 1     0 x x
    MiniQ 8-X     0 x x
    Plamsid X 100ng in total   49 2.0408163265 5.9591836735

    Incubate the digestion solution at 37 degrees for 1h

    Heat shock 80 °C for 20min to stop the enzymatic reaction


    The ligation solution should contain the following parts (uL): see table 8

    The amount of gene should have equal molar weight to the plasmid

    Using the NEB calculator.

    Table 8: Ligation mixture

    Plasmid back bone 2  
    gene 1 X \<3
    gene 2 Y \<3
    T4 ligase 0.5  
    T4 ligase buffer 1  
    MiniQ 6.5-X-Y  

    Leave the solution at room temperature and react for 1.5h

    Heatshock at 80 °C for 20 min

    Use 5uL (half) of the ligation solution to transform the bacteria.

  • Co-transformation

    Prepare dilutions of the plasmids with a concentration of 2 ng/µL

    Switch on the water bath and set temperature at 42 °C. Also, turn on the heat/shaking-block and set up to 37 °C

    Load a bucket with ice from the ice machine

    Take the bacterial cells and SOC (Super optimal broth with catabolite repression) out of the -80 °C freezer. Transfer the cells directly to ice. Do not touch the bottom of the tube that contains the cells.

    Thaw the cells on ice for ~5 minutes

    Add 1 μL of each plasmid into 20 μL bacteria. Mix well. Make sure you work near the Bunsen burner flame

    Leave the cells on ice for 5 minutes

    Heat-shock the cells for 30 seconds (exactly!) at 42°C

    Return the cells directly to ice for 2 minutes

    Add 80 μL of SOC solution to the bacteria

    Incubate for 60 minutes at 37 °C and 300 rpm

    Dry agar plate, supplemented with Spectinomycin(25 µg/mL) and Kanamycin(30 µg/mL) in the 37 °C incubators. Place the plate upside down and slightly opened.

    Plating the cells on agar plate

    Take the dried agar plate out of the 37 °C incubators

    Label the bottom of the plates

    Open an agar plate in close proximity of the Bunsen burner flame

    Pipette the cells (100 μL) on the plate

    Sterilize the Drigalski spatula by burning the alcohol on it, shortly let it cool down

    Spread the cells on the plate using the sterile spatula

    Transfer the agar plate to the 37°C incubator

    Place the plate upside down, closed • Let the cells grow on the plate overnight.

  • E. coli transformation

    This protocol describes how to transform competent E. coli.__Protocol is adapted from Marjolein Crooijmans, iGEM 2018

    1. Competent E.coli cells,
    2. Plasmid DNA or Ligation reaction
    3. Ice
    4. Waterbath at 42°C
    5. LB medium
    6. LB plates with appropriate antibiotics
      1. Ampicillin (100 µg/mL)
      2. Kanamycin: (50 µg/mL)
      3. Chlorampenicol: (25 µg/mL)
      4. Spectinomycin: (50 µg/mL)

    Thaw 50 ul competent cells on ice

    Add 10 ul ligation mix DNA and mix carefully

    30-60 min on ice (30 min is completely fine)

    Incubate 90s 42oC (heat-shock) and immediately place back on ice

    Add 1 ml LB medium (do 400 uL instead)

    Incubate 1.5 hour at 37OC without shaking (1 hour is also fine)

    Plate on LB agar plates with appropriate antibiotics

    Incubate overnight at 37OC

  • PCR-based cloning

    Ligating promoters in front of the toxins with PCR-based protocol.

    PCR reaction


    Forward primer

    Reverse primer

    Taq polymerase

    Taq buffer

    Table 9: PCR mixture

    DNA 1 ul
    Promoter 1 ul
    Forward primer 2 ul
    Reverse primer 2 ul
    Taq DNA buffer 2 ul
    Taq polymerase 0.25 ul
    MilliQ 16.75 ul
    Total 25 ul

    Table 10. PCR gradient program

    Phase Repetitions Temperature (C) Time (s) Notes
    1 1 time 95 30  
    2 15 times 95 30  
    2   68 30 -0.6 C / cycle
    2   72 60  
    3 20 times 95 30  
    3   59 30  
    3   72 60  
    4 1 time 72 300  
    4 1 time 4 infinity  
  • Competent cell CFU test

    1. Shaker
    2. Consumable
      1. Eppendorf tube *9
      2. LB medium
      3. Ice
    3. Competent cells
    4. Plasmid DNA

    Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

    Do triplicates (3 each) of each concentration if possible, so you can calculate an average colony yield.

    Spin down the DNA tubes containing your controls to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient.

    Pipet DNA into each microcentrifuge tube. Recommend final concentration in 1mL medium is 0.01ng/mL, 0.05ng/mL and 0.1ng/mL

    Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

    Incubate on ice for 30 minutes.

    Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

    Heat-shock the cells by placing into the waterbath for 45 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

    Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes.

    Add 950 µL of SOC media per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

    Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.

    Pipet 100 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.

    Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.

    Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg)

    Note: The measurement "Amount of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:

    Amount of DNA plated (ng) = Volume DNA added (1 µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100 µL) / total reaction volume (1000 µL)]

  • Electroporation protocol

    This method of competent cell preparation follows the TSS preparation described by Chung & Miller. Chung, C. T., & Miller, R. H. (1993). Preparation and storage of competent Escherichia coli cells. Methods in enzymology, 218, 621–627.

    1. Bacteria
      1. E. coli strains of choice, for example DH5α
    2. Consumables
      1. dimethyl sulfoxide (DMSO)
      2. polyethylene glycol (PEG)
      3. Ice
      4. Liquid nitrogen
      5. 2 M MgCl2 (1.94 g in 100mL)
      6. LB medium
    3. Equipment
      1. 250 ml Erlenmeyer
      2. Centrifuge
      3. Shaking incubator
      4. Spectrophotometer
      5. 50 mL tubes

    Table 11. TSS medium (pH 6.5)

    Component Stock (M) Amount Unit Final concentration  
    MgCl2 1 2.5 ml 50 mM
    DMSO - 2.5 ml 5 %
    PEG -6000 - 5 g 10 %
    LB   up to 42.5 ml - -

    Day 1

    Grow preculture LB (10 ml) at 37OC overnight

    Prewarm 500 ml medium at 37OC

    Day 2

    Inoculate 2x 2.5 ml preculture in 250 ml prewarmed LB medium

    Grow 2-2.5 hours at 37OC to OD600 = 0.3-0.4 (not \>0.4)

    Pellet cells by centrifugation (10min, 4000rpm centrifuge 5 degrees)

    Resuspend pellet in 5 ml cold (!) TSS medium

    Divide suspension in 50 ul portions

    Immediately cool down each tube in liquid nitrogen

    Store at -80 °C

    Note: This protocol does not require the administration of a heat shock: incubating the cells with the DNA on ice is enough to obtain high transformation rates. According to the article, administering a heat shock may lower transformation efficiencies.

  • E. coli competent cells TSS method

    Preparation of LB agar plates

    1. Consumables
      1. LB medium 25 g/L
      2. Bacterial Agar 15g/L
    2. Antibiotic stocks (1000X)
      1. Ampicillin (100 mg/mL)
      2. Kanamycin: (50 mg/mL)
      3. Chlorampenicol: (25 mg/mL)
      4. Spectinomycin: (50 mg/mL)
    3. Equipment
      1. Autoclave
      2. Bunsen burner
      3. Incubator

    Microwave solidified LB agar bottles until they are almost boiling or use freshly autoclaved LB agar.

    Let the agar cool until you can hold it in your hands for 10 seconds

    Optional: For selection plates add for 1 mlof LB agar,1 µL of the antibiotic stock

    Pour 25 mL LB agar in a petri-dish and let the agar solidify.

    Label the Petri dishes with the appropriate color corresponding to the antibiotics used!

    Store the plates upside down at 5°C

  • Standard protocol for pouring plates

    Making your own electrocompetent cells



    1. 2% tryptone
    2. 5% yeast extract
    3. 10 mM NaCl
    4. 5 mM KCl
    5. 10 mM MgCl2
    6. 10 mM MgSO4


    - SOB + 20 mM glucose

    Appropriate Antibiotics for Your Application.

    Antibiotics for Plasmid selection.

    Antibiotic Working Concentration

    1. Ampicillin 100 µg/ml
    2. Carbenicillin 100 µg/ml
    3. Chloramphenicol 33 µg/ml
    4. Kanamycin 30 µg/ml
    5. Streptomycin 25 µg/ml
    6. Tetracycline 15 µg/ml

    Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol). Procedure (for 2, 250 ml cultures)

    1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37°C and 250 rpm.
    2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. Shake at 37°C and 250 rpm until the cultures reached an OD600 of 0.5-0.7. Be sure to turn on the centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
    3. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
    4. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
    5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
    6. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. If necessary, adjust the final volume of cells so that the OD600 is within the range 210-270.
    7. At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the cultures, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80 °C freezer. The cultures should be good for \>6 months.

    Electroporation Protocol

    1. The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.
    2. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
    3. Turn on electroporator and set to 1.7-2.5 kV (optimize for strain), 200 ohms and 25 µF. Place recovery SOC in 37°C water bath. Pre-warm LB-antibiotic plates at 37°C. Thaw cells on ice for 10 min or use freshly made cells. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice .
    4. Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
    5. Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
    6. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don't hold the button down).
    7. Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
    8. Rotate in the 37°C incubator for 1 h.
    9. Make appropriate dilutions. When using 10 pg of DNA, make two dilutions: Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution) Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution) Incubate overnight at 37°C. Calculation: If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml) cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)