Wet Lab>Low-temperature Release System

Outline


    The recombinant protein expression system has contributed greatly to structural and functional analyses of proteins and their applications. Recombinant protein expression systems are usually employed for the overproduction of proteins. Nevertheless, there are still many proteins whose overproduction by a recombinant protein expression system is difficult. These include proteins with low stability, proteins that are toxic to the host, and proteins that tend to form inclusion bodies. To overcome this problem, a temperature-dependent genetic circuit was designed and constructed in E. coli. A specific ribosome binding site sequence BBa_K115002 which has high translation activity at high temperature(>37 C) and low translation activity at room temperature was used to design the temperature-dependent genetic circuit in E. coli, and the green fluorescent protein (GFP) was used as the reporter protein. We analyzed fluorescence intensity and the OD ratio during E. coli growth at log phase and stationary phase at 25C, 30C, 37 C and 40C. Our experimental results indicated that the low temperature release system is function as our design. The expression level of the target protein can be control by the incubation temperature of the host cell with this temperature-dependent genetic circuit.
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Fig. 1: The low temperature release system design in the Mosquito Intelligent Terminator. This temperature-dependent circuit has high translational activity at high temperature (>37° C) and low translation activity at room temperature.

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Component Descriptions

    1. In strand A, the constitutive promoter (BBa_J23101) transcripts the mRNA of tetR (BBa_C0040) constitutively, and the production rate of tetR protein is controlled by the translational activity of a temperature-sensitive RBS BBa_K115002. Thus, strand A can control the expression level of strand B by the host cell's incubation temperature.

   2. The concentration of tetR repressor controls the transcriptional activity of Ptet promoter (BBa_R0040) to produce GFP protein.

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Principle and Mechanism

   The low temperature release system has two statuses:
      1. Reproduction status at 37°C in laboratory
      2. Downstream protein release status at less than 37°C.

1. Reproduction status at 37°C in laboratory

   When bacterium grows in 37°C, the constitutive promoter transcript tetR repressor inactivates the promoter Ptet (BBa_R0040) on strand B, subsequently downstream protein (GFP or other protein) is not transcribed to restrict the host growth (Fig. 2). In this condition, almost none of the green fluorescent protein can be detected. This design allows us to amplify the terminators without any potential limiting factors. Image description

Fig. 2:The reproduction status when bacterium grows at higher than 37°C.

 

2. Downstream protein release status at less than 37°C.

   When the E. coli are released in the environment at room temperature (<37°C), the translational activity of temperature-sensitive RBS BBa_K115002 is repress by low temperature. Thus, the promoter Ptet promoter transcribes the downstream protein (GFP) constitutively (Fig. 3). Image description

Fig. 3: Downstream protein release status at less than 37°C

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Procedures

Flow Cytometry Methods

(I) Transformation of BBa_ K332032 into E. coli EPI300

  1. Thaw EPI300 E. coli cells and BBa_ K332032 plasmid on ice.
  2. Transfer 1μL of plasmid and 100μL of cells to a pre-chilled microcentrifuge tube.
  3. Incubate on ice for 5 minutes.
  4. Transfer the tubes to 42°C for 1 minute.
  5. Transfer the tubes back to ice and cool for 5 minutes.
  6. Add 200μL lysogeny broth to each tube.
  7. Recover the cells by incubating at 37°C for 30 minutes.
  8. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 µg/ml kanamycin.
  9. Incubate the cultures at 37°C overnight.

(II) Preparation of M9 minimal media

  1. Dissolve 1.695g Na2HPO4, 0.75 g KH2PO4, 0.125g NaCl, 0.25 g NH4Cl and 0.5 g Casamino acid in 250 mL double distilled water (ddH2O).
  2. Sterilize the solution at 121°C for 30 minutes by using moist heat sterilization method.

(III) Flow cytometry

  1. Pick one single colony off the transformation plate of E. coli EPI300. Dilute the bacteria with 20μL ddH2O.
  2. Add 4μL diluted bacteria into 4mL M9 minimal media supplemented with 0.2% casamino acids, 1 mM MgSO4, 2 μM thiamine, 10 mM glucose, and 50ng/ml Kanamycin. (For all of the measurements, cells were grown in M9 minimal media supplemented with 0.2% casamino acids, 1 mM MgSO4, 2 μM thiamine, 10 mM glucose, and 50 ng/ml Kanamycin antibiotic.)
  3. Incubate at 37°C overnight on a shaker at 200 rpm.
  4. Prepare 4 tubes, since we will culture these samples at 4 different temperatures. To each tube, add 4μL of the above-mentioned culture into 4mL fresh M9 media supplemented with 0.2% casamino acids, 1 mM MgSO4, 2 μM thiamine, 10 mM glucose, and 50 ng/ml Kanamycin.
  5. Incubate the tubes at 37°C with shaking at 200 rpm until an optical density of 0.1 is reached.
  6. Transfer the tubes to their corresponding temperatures, including 25°C, 30°C, 37°C, and 40°C. Use flow cytometry to measure the fluorescence and observe the changing patterns for more than 7 hours.

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Result

 

Above 37°

Below 37°

Bacterial growth rate

Accelerated

Slowed

RBS K115002 (37 °C induced)

Activated

Inactivated

tetR

Activated

Inactivated

pTet(inhibited by tetR)

Inhibited

Not inhibited

GFP (or cry gene)

Inhibited

Not inhibited


  We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37° C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37° C induced RBS (K115002), the tetR gene and terminators. We will call this part the "Regulator"(Figure1).

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   Second, we combine pTet Promoter, RBS B0034, the crystal protein gene and terminators. We call this part the "Production"(Figure2)

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  We use the "Regulator"> to maintain a stable growth rate of bacteria while inhibiting crystal protein protection by way of the tetR gene. Bacteria growth, particularly vector mutation rate and growth rate, is highly dependent on amplification conditions, thus crystal protein production is off during the incubation/amplification step. Because the tetR gene of the "Regulator" can block the pTet promoter of "Production", we achieve a self-regulating, temperature dependent system that monitors both bacteria growth and protein production. We speculate that when the "Regulator" and "Production" sections are combined, we can achieve a high bacteria growth rate and close to no crystal protein producing rate at T>37° C, and low bacteria growth rate and high crystal protein producing rate at T<37° C.(Figure3)

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  To test the efficiency of the temperature induced RBS and the interaction between tetR gene and ptet promoter, we replaced the crystal protein gene with GFP (green fluorescence protein) gene. In this scenario, GFP will simulate the crystal protein's production, as it is easily detected by flow cytometry.(See Cytometry figure)

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   To conclude, we have verified that our strategy works. Figure 4 demonstrates the regulation of GFP in a temperature dependent fashion, as it is evident the mean GFP values are significantly lower at T>37° C. We expect our circuit design to allow steady bacteria growth and inhibit crystal protein at T>37° C, and high protein production and low bacteria growth rate at T<37° C

 

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