Part:BBa_K1033001

4-coumarate ligase (4CL) with RBS

4-coumarate ligase (4CL) is an enzyme that catalyses the reaction from p-coumaric acid to 4-coumaroyl-coenzyme A (4-coumaryl-CoA). This enzyme is derived from the plant arabidhopsis thaliana, but exists in many other plants. [1]

In our project, we have been using it together with stilbene synthase, that produces resveratrol with the help of this enzyme.

Applications With the help of this enzyme one can produce 4-coumaryl-CoA which is an important precursor in many metabolic pathways. For example, it can be used to produce resveratrol with stilbense synthase. [2]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1108
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1675
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1307
  

iGEM 2020 QHFZ-China, new documentation (For Bronze)

Author: Yixian Yang

     We measured BBa_J23100, BBa_J23107 and BBa_J23109 as a strong, moderate and weak promoter respectively in 2020. For all the experiments below, we use E. coli BL21(DE3) strain.

Part 1: Measurement with a reprter, sfGFP

Description

    First, we measured the strength of the promoter by sfGFP BBa_K3457015.

Protocol

    The gene circuit we used is as below:

Figure 1. The Schematic cartoon of the DNA construct to test J23100 / J23107 / J23109 with sfGFP.

    The protocol is as below:
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
(3) The bacteria solution was centrifuged and the LB medium was removed. Then the bacteria was resuspended by PBS. 100 μL such solution was put into a well of a 96-well palte. The GFP fluorescence and OD₆₀₀ were detected by a microplate readers (Bio-Teck). The parameters are: exciting light: 488 nm, light reception: 520 nm, gain: 50.
(4) The value of PBS was deducted from the result above. GFP / OD₆₀₀ was calculated.

Result

Figure 2. sfGFP was expressed with J23100 / J23107 / J23109.

    We set the strehgth of J23109 as 1. The relative strengths of J23107 and J23109 were 4.4 and 12.0. Though they are not the same as the data at the top of this page, they worked well anb the strength order of the three promoters was accordance was consistent with other people's data. The difference may owe to the certain gene circuit and protocol.

Part 2: Measurement with CHAS 106094

Description

    Second, we measured the strength of the promoter by CAHS 106094 BBa_K3457012. This year, we used CAHS 106094 to protect bacteria from freeze-drying and dry storage. We used different promoters to adjust the expression level of CAHS 106094, to study the relationship between the survival rate and CAHS 106094 expression level.

Protocol

    The gene circuit we used is as below:

Figure 3. The Schematic cartoon of the DNA construct to test J23100 / J23107 / J23109 with CAHS 106094.

    The protocol is as below:

Figure 4. Experiment protocol.

【Day 1】Induction culture
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the experiment.
(2) Use spectrophotometer to measure the OD₆₀₀ of the bacteria solution, OD₆₀₀ = 1 equals to 10⁹ cells. If the OD₆₀₀ value is between 0.1 and 1, There is a linear relationship between OD₆₀₀ and bacterial density. Calculate the volume of bacterial solution for 10⁹ cells by using the formula V = 100 / (OD₆₀₀ × Dilution ratio).
(3) Take out a measured amount of 10⁹ cells and centrifuge it at 8000 rpm for 3 min. Then pour out the supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum pump to dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria solution on the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after several gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the colone number on the LB plate, then compare the results between each group.

Result

Figure 5. The Cfu of bacteria expressing CAHS 106094 after freeze-drying with J23100 / J23107 / J23109.

    As expected, J23100 is the strongest promoter and it gave the best survival rate. J23107 is the second and J23109 seemed too weak to express enough CAHS 106094. In conclusion, J23100 and J23107 is effective in this situation, but J23109 is not.