Team:Edinburgh UG/Measurement



As a standardized toolkit for site-specific recombination, SMORE complies with the principle of biobrick and synthetic biology, emphasizing on accurate and repeatable measurements of our parts. This is why we have developed simple protocols and specific biobricks to quantify the activity of recombination, in terms of both efficiency and orthogonality.

This protocol involves i) transferring the recombinase generator from pSB1C3 to pSB4K5; ii) transformation of T7 polymerase expressing E. coli strain with recombinase generator in pSB4K5; iii) make competent cells out of the recombinase-carrying E. coli; and iv) transformation of recombinase-carrying E. coli with the appropriate measurement device. The entire experiment takes 7 days.


Material needed

  1. Escherichia coli strain for cloning purpose (e.g. TOP10, DH5α)
  2. Any T7 polymerase expressing E. coli strain (e.g. BL21 (DE3))
  3. Biobricks: pSB4K5 plasmid backbone
  4. Biobricks: T7-LacO-regulated Cre/Dre/VCre/SCre/Vika recombinase generators (BBa_K2406080, BBa_K2406081, BBa_K2406082, BBa_K2406083, BBa_K2406084)
  5. Biobricks: Measurement device (BBa_K2406051, BBa_K2406053, BBa_K2406055, BBa_K2406057, BBa_K2406059, BBa_K2406061, BBa_K2406063, BBa_K2406065, BBa_K2406067, BBa_K2406069, BBa_K2406071, BBa_K2406073, BBa_K2406075, BBa_K2406077, BBa_K2406079)
  6. Chloramphenical (stock of 25 mg/mL in EtOH
  7. Kanamycin (stock of 50 mg/mL in dH2O)
  8. IPTG (stock of 0.1 M in dH2O)
  9. LB (Luria Bertani) media and LB agar
  10. MgCl2 (Autoclaved, 0.1 M)
  11. CaCl2 (Autoclaved, 0.1 M)
  12. 100% glycerol
  13. Standard biobrick restriction enzymes – EcoRI and SpeI, and their associated enzyme buffer
  14. T4 DNA ligase and its associated enzyme buffer
  15. Miniprep kit
  16. 50 mL falcon tubes
  17. 1.5 mL eppendorf tubes
  18. Cuvettes
  19. Erlenmeyer flask (250 mL or larger)
  20. Petri dishes
  21. 96-well plates (black ,with flat and transparent bottom)
  22. Pipettes
  23. Spectrophotometer
  24. Plate reader
  25. Water bath and/or heat block
  26. Ice and ice buckets
  27. Dry ice or liquid nitrogen
  28. Microcentrifuge (for eppendorf tubes)
  29. Centrifuge (for falcon tubes)
  30. Incubator at 37ºC


Day 1:

  1. Transform E. coli DH5α with the following inducible recombinase generators:
    1. T7-LacO-Cre (BBa_K2406080)
    2. T7-LacO-Dre (BBa_K2406081)
    3. T7-LacO-Vika (BBa_K2406082)
    4. T7-LacO-VCre (BBa_K2406083)
    5. T7-LacO-SCre (BBa_K2406084)

    Plate on LB agar with chloramphenicol
  2. Transform E. coli DH5α with the following measurement constructs:
    1. Rox-term-Rox (BBa_K2406051)
    2. Vox-term-Vox (BBa_K2406053)
    3. Lox-term-Lox (BBa_K2406055
    4. Vlox-term-Lox (BBa_K2406057)
    5. Vlox-term-Vlox (BBa_K2406059)
    6. Slox-term-Lox (BBa_K2406061)
    7. Slox-term-Vlox (BBa_K2406063)
    8. Slox-term-Slox (BBa_K2406065)
    9. Vox-term-Vlox (BBa_K2406067)
    10. Vox-term-Slox (BBa_K2406069)
    11. Rox-term-Slox (BBa_K2406071)
    12. Rox-term-Vox (BBa_K2406073)
    13. Lox-term-Vox (BBa_K2406075)
    14. Vlox-term-Rox (BBa_K2406077)
    15. Lox-term-Rox (BBa_K2406079)

    Plate on LB agar with chloramphenicol
  3. If the users do not have miniprepped DNA of pSB4K5 backbone, transform E. coli DH5α with it as well, and plate on LB agar with kanamycin.
  4. Incubate all the LB agar plates with transformants overnight at 37ºC.

Day 2:

  1. Inoculate the twenty transformed parts (pSB4K5 too if done) with 5 mL LB broth with chloramphenicol. Shake at 220 rpm, 37ºC overnight (16 – 18 hours)

Day 3:

  1. Miniprep the twenty parts (pSB4K5 too if done) using the miniprep kit.
  2. Reserve the fifteen measurement devices for later use. For the inducible recombinase generator, mix its miniprep with the miniprep of pSB4K5, and perform restriction digest and heat inactivation with EcoRI and SpeI according to the recommendation by supplier.
  3. Add T4 DNA ligase and its buffer to the heat-inactivated reaction mix (make sure that the T4 DNA ligase is not incompatible with the previous buffer). Incubate according to the supplier’s recommendation.
  4. Transform the ligated products (a total of five, one for each recombinases) into E. coli BL21 (DE3). Plate on LB agar with kanamycin.

Day 4:

  1. Pick out a white colony from each of the five transformations done yesterday. Inoculate the white colony in 10 mL of LB broth + kanamycin overnight (220 rpm, 37ºC).

Day 5:

  1. Inoculate 100 mL of LB broth + kanamycin with 1 mL of overnight culture. Shake at 220 rpm, 37ºC for approximately 2 hours, or until the OD600 reaches 0.3 – 0.6 (absorbance at 600 nm).
  2. Transfer the culture to two 50mL falcon tubes and leave on ice for 30 minutes.
  3. Transfer at least 150 mL of 0.1M MgCl2 and 150 mL of 0.1M CaCl2 onto ice.
  4. Prepare 20 mL of CaCl2/Glycerol solution by mixing 17mL of 0.1M CaCl2 with 3mL of 100% glycerol.
  5. Centrifuge at 4000x g for 5 minutes at 4ºC.
  6. Pour out the supernatant and resuspend the pellet gently with 25 mL ice cold 0.1M MgCl2.
  7. Incubate on ice for 30 minutes.
  8. Centrifuge at 4000x g for 5 minutes at 4ºC.
  9. Pour out the supernatant and resuspend the pellet gently with 25 mL ice cold 0.1M CaCl2.
  10. Incubate on ice for 30 minutes.
  11. Centrifuge at 4000x g for 5 minutes at 4ºC
  12. Resuspend the pellet gently with 1.25 mL of CaCl2/Glycerol solution prepared earlier.
  13. Aliquot 100 μL of cell resuspension into eppendorf tubes.
  14. Flash freeze the aliquots on dry ice or liquid nitrogen.
  15. Store the recombinase-expressing competent cells in -80ºC freezer.

Day 6:

  1. Thaw the recombinase-expressing competent cells on ice. You will need five tubes for each recombinase.
  2. Transform the measurement devices into the competent cells according to this table:
    Competent cells Measurement devices to transform
    1. Lox-term-Lox (BBa_K2406055)
    2. Vlox-term-Lox (BBa_K2406057)
    3. Slox-term-Lox (BBa_K2406061)
    4. Lox-term-Vox (BBa_K2406075)
    5. Lox-term-Rox (BBa_K2406079)
    1. Rox-term-Rox (BBa_K2406051)
    2. Rox-term-Slox (BBa_K2406071)
    3. Rox-term-Vox (BBa_K2406073)
    4. Vlox-term-Rox (BBa_K2406077)
    5. Lox-term-Rox (BBa_K2406079)
    1. Vox-term-Vox (BBa_K2406053)
    2. Vox-term-Vlox (BBa_K2406067)
    3. Vox-term-Slox (BBa_K2406069)
    4. Rox-term-Slox (BBa_K2406071)
    5. Lox-term-Vox (BBa_K2406075)
    1. Vlox-term-Lox (BBa_K2406057)
    2. Slox-term-Vlox (BBa_K2406063)
    3. Vox-term-Vlox (BBa_K2406067)
    4. Vlox-term-Rox (BBa_K2406077)
    5. Vlox-term-Vlox (BBa_K2406059)
    1. Slox-term-Slox (BBa_K2406065)
    2. Slox-term-Lox (BBa_K2406061)
    3. Slox-term-Vlox (BBa_K2406063)
    4. Vox-term-Slox (BBa_K2406069)
    5. Rox-term-Slox (BBa_K2406071)

Day 7:

  1. Inoculate a colony from each of the transformation into 5 mL of LB broth + chloramphenicol and kanamycin in the morning.
    *With only 96 wells in each plate, it is recommended to split the 25 transformations into several experiments, inoculate only a portion of the transformants each time.
  2. When the OD600 reaches 0.3 – 0.6, dilute the cultures with LB broth + chloramphenicol and kanamycin until all the cultures have similar OD600, between 0.1 – 0.2.
  3. Aliquot 100 μL of the diluted cultures into the 96-well plates. Reserve a column for loading the blank (LB Broth with chloramphenicol and kanamycin, without any E. coli). To half of the cultures, add 0.5 μL of 0.1M IPTG to reach a final concentration of about 500 μM.
  4. Seal the 96-well plate and put into the plate reader. Measure the RFP fluorescence intensity every 30 minutes over 48 hours with the following conditions:
    • Excitation: 584 nm
    • Emission: 610 nm
    • Shaking frequency: 300 rpm
    • Shaking mode: double orbital
    • Incubation temperature: 37ºC


Results are summarized below. We have obtained quantitative results regarding the cross-reactivity of our four recombinases: Dre, Vika, VCre, and SCre. Our devices detected some unexpected cross-reactivity. For example, VCre and SCre were shown to recognize one another’s target site. Below is a “heat map” of every recombinases activity with respect to every measurement device tested. Note grey corresponds to constructs that were not tested due to time constraints. Also included is a graph demonstrating observed fluorescence output over time for every single measurement taken by our team.

Heat map showing describing the orthogonality of different recombinase systems in E. coli. Percentage of orthogonality is normalised with Rox-Rox in Dre being 100%.


The results show that are measurement constructs function and are able to detect cross-reactivity between two recombinases. Indeed, observed sensitivity of our devices was so high that we demonstrated significant cross-reactivity between SCre and VCre, two recombinases originally reported as orthogonal [1]. Furthermore, we demonstrate that there is minimal cross-reactivity between Dre [2], Vika [3], and Cre, indicating that these recombinases can be used to catalyse distinct recombination events within one cell.

Our measurement protocol is also very repeatable. Any team wishing to carry out their own measurement using our parts could simply order the relevant parts from the registry and follow our above protocol. Also, the principle behind these measurement devices is useful for other projects. For example, if another team wanted to utilize Gin and Tn3 invertases [4][5] in one cell, they would be wise to first test if the two systems were orthogonal. They could achieve this by following our design plan and our wet-lab protocol. Our project would simplify the process of designing constructs through our web-based support tool: our oligo designer. Our recombinase target sites could be tricky to design because they contain repetitive sequences that cannot be chemically synthesized.

In conclusion, we have developed a robust tool for assessing recombinase activity. Our measurement devices provided invaluable insight into our own project by distinguishing which of our recombinases were orthogonal and which would cross-react. The devices we have produced are valuable to the iGEM community as a whole due to our detailed protocol, submission of standardized measurement devices, generalizable platform, and web-based support for designing elements of the devices.


[1] Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49.

[2] Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515

[3] Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

[4] Maeser, S. and Kahmann, R. 1991 “The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts.” Molecular Genetics and Genomics 230(1-2):170-176

[5] Olorunniji, F.J. and Stark, W.M. 2010 “Catalysis of site-specific recombination by Tn3 resolvase”. Biochemical Society Transactions 38(2):417-421