Part:BBa_K3040501

Lipase-Pseudomonas sp 7323.

Lipase from Pseudomonas sp 7323

Description

Lipase from Antarctic cold-tolerant Pseudomonas sp. 7323 can catalyze the hydrolysis of triacylgly-cerols to glycerol and monoacyl-glycerols. We utilize the characteristic of lipase-having different catalytic activity under different temperature, to sense the temperature change. Lipase A reaches its catalytic activity peak at about 30℃, and decreases as temperature rise or drop. Furthermore, to purify our lipase A in an easier way, we design a His-tag sequence behind the lipase A.

Result

Prove our vectors successfully constructed

The cold-adapted lipase A is from an Antarctic deep sea psychrotrophic bacterium Pseudomonas sp. 7323. Lipases are glycerol ester hydrolases that are able to hydrolyze ester to free fatty acid and glycerol. With overexpression of Lip A, the bacteria are able to produce different concentration of fatty acid in different temperature.

We get the sequence of Lip A from NCBI. In order to check the expression of Lip A in cells and facilitate the purification of this protein, we attached the 6 X His tag on the C-terminal of this protein. This part was inserted into the iGEM provided expression vector psB1C3 through the restriction site EcoRI and SpeI (Fig1).

Figure 1 Construction of expression vector pSB1C3-LipA-6X His-tag. The insert sequence is flanked by EcoRI and SpeI restriction site.

This recombinant plasmid was further screened by ampicilin selection, colony PCR in the cloning E. coli, DH5α (Fig. 2) and the digestion of miniprep product (Fig 3). From those result, we can prove that the Lip A sequence synthesized by IDT was successfully integrated into the cloning vector psB1C3.

Figure 2 DNA electrophoresis with 1.32% gel was performed to screen the positive recombinant. The plasmid constructed was 4176bp and the predicted PCR result should be 2420bp (flanked by the VF2 and VR primer). Lane 1: DNA loading marker, Lane 2-7: VF2 and VR PCR product).

Figure 3 DNA electrophoresis with 1.32% gel was performed to screen the positive recombinant. The plasmid extracted was digested with EcoRI and SpeI. The digested part (R0010-Lip A-6X His tag) should be 2129bp. Lane 1: DNA loading marker, Lane 2 to 5: Plasmid digested, Lane 6: J04450 control. The plasmid should be digested into 2 parts, one is R0010-Lip A-6X His tag, the other one is the psB1C3 backbone, both parts are about 2200bp. Thus, there will be only one band on the lane.

Prove lipase successfully produced

We have transformed E. coli BL21 strain with pSB1C3-LipA-His tag construct which has been previously proved succeed. The positive transformants were screened with ampicillins and colony PCR. We liquid cultured the cells and collected after 16 hours. After washing with PBS, we lysed the cells by using lysis buffer (please refer to the Experiment protocol) to get the protein lysate. Western Blot has been performed to check the expression of Lip A by using antibody against His tag.

Figure 4 Western Blot analysis of total protein extracted from BL21 transformed with pSB1C3-Lip A-6X His tag by using antibody against His tag. Lane 1: BL21 control with no plasmid transform. Lane 2 and 3: Protein loading marker. Lane 4 to 13: BL21 transformed with our construct. According to the information on UniProt, Lip A from Pseudomonas sp.7323 was reported to be 64.555 kDa.

Prove enzymes are functional

In order to verify the functionality of Lip A expressed, we have done a functional assay with the protein extract from BL21 by sonication. To determine the lipase activity, we utilized a spectrometry-based method by using 4-nitrophenyl decanoate as substrate. The amount of 4-nitrophenol hydrolyzed and released was determined spectrophotometrically at 405nm. We compared the fluorescence and found that the change in fluorescence is greater in the protein in BL21 expressed Lip A compared to control (Fig. 5).

Figure 5 Lipase activity assay was performed to analyze the function of lipase A. The protein was first incubated at 30°c for 30 minutes at pH9.0. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm 30°c in continuous duration (0, 20, 40, 60 mins). The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

Enzymes activity detection

Since our goal is to build a precise thermal-tunable promoter with dynamic range of gene transcription, the lipase activity in variety of temperature is very important to us. Hence, we evaluated the lipase activity in different temperature and the curve illustrated fitted to what reported in previous research of Lip A of Pseudomonas sp.7323 (Figure 6 and 7).

Figure 6 Lipase activity assay analysis was performed to check the activity of lipase at different temperature varies with time at pH9.0. The protein was first incubated at the experiment temperature (10, 20, 25, 30 and 40°c) for 30 minutes. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm in continuous duration (0, 20, 40, 60 mins) at the temperature required. The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

Figure 7 Lipase activity assay analysis was performed to check the activity of lipase at different temperature for 40 minutes at pH9.0. 40 minutes data was chosen because this time duration is best fitted to the activity curve reported previously in the paper. Thus, our lipase is proved to be functional.

In conclusion, lipA is the functional and the activity fits our expectation. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 40
  Illegal suffix found in sequence at 2168
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 40
  Illegal SpeI site found at 2169
  Illegal PstI site found at 2183
  Illegal NotI site found at 46
  Illegal NotI site found at 2176
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 40
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 40
  Illegal suffix found in sequence at 2169
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 40
  Illegal XbaI site found at 55
  Illegal SpeI site found at 2169
  Illegal PstI site found at 2183
  Illegal NgoMIV site found at 436
  Illegal NgoMIV site found at 548
  Illegal NgoMIV site found at 1583
  
• 1000
  COMPATIBLE WITH RFC[1000]