Team:Hong Kong UCCKE/Contribution
Contribution
Characterization of BBa_K523006 designed by Team Edinburgh in 2011
Purpose
Team Edinburgh conducted 2 iodine assays to compare the catalytic activity of BBa_K23006 colonies with a negative control and a known control PlacLacZ-bglX, which proved the ability of malS to degrade starch. Therefore, we undertook the experiment by comparing the catalytic activity of BBa_K23006 on starch with a positive control, 1% amylase solution.
Iodine Assay
Design
To compare the catalytic activity of α-amylase(malS) with 1% amylase on starch, we carried out this assay to test their effect on starch(delete). The catalytic activity is measured by the diameter of the clear zone in cm formed by the two testing samples under iodine solution on each day.
Below is the protocol for our assay:
Materials
| Materials | Amount |
|---|---|
| Agar plate(with LB, starch, antibiotics) | x15 |
| Clones of α-amylase | |
| 1% amylase | |
| Micro-pipette(20 ul) | x1 |
| Box of tips | x1 |
| Marker | x1 |
| Ruler | x1 |
Procedures
- Use marker to draw a dotted line and cut the agar plate into 2 parts with the help of a ruler.
- Draw a straight line on both parts with a distance 2 cm away from the dotted line.
- Label A for amylase and B for Clones of a-amylase on the sides on the Agar plate respectively.
- Label day1, day2 and day3 on three set of three Agar plate respectively.
- Draw a cross on both sides on the straight line
- Conduct a sterile space for the experiment below
- Use Micro-pipette (20ul) to transfer a spot of 10ul 1% amylase on the cross on the A side on the Agar plate.
- Use Micro-pipette to transfer a spot of Clone of a-amylase on the cross of on the B side on the Agar plate.
- Repeat step 7-8 for the five clone of a-amylase on each Agar plate
- Put all agar plate in the incubator of 37 celcius degree.
- Take results on day1,2,3 respectively by preforming the iodine test
Iodine test
Materials
| Materials | Amount |
|---|---|
| Iodine solution | x1 |
| Dropper | x1 |
| Agar plate | x3 |
| White tile | x1 |
| Marker | x1 |
| Ruler | x1 |
Procedures
- Take the agar plates out from the incubator.
- Use a dropper transferring the iodine solution to the Agar plate with the solution just fully cover the Agar surface.
- Cover the plate and shake the plate gently.
- Observe the clear zone occurred .
- Measure and record the diameter of clear zone on agar plate with a ruler for both A & B side on each sample.
- Use a marker to mark the shape and size of the clear zone on its plate cover.
Results
Day 1*
Day 2*
Day 3*
*1% amylase solution (+ control) added on the top, clone of BBa_K23006 in the bottom
Graph 1: The diameter of clear zone of α-amylase and 1% amylase
The assays lasted for 3 days, results for the iodine test is collected every day.We took the results from 5 α-amylase clone and 1% Amylase. In 3 days, a growth in clear zone and halos around the bacteria with malS clone is observed under iodine solution , which showed malS positive in the degradation of starch. From the graph above, 1% amylase showed a greater starting starch degradation amount with a longer clear zone diameter.Yet, it has a constant diameter of clear zone which seemingly levels off in day 3, and suggests that the degradation rate slows down with time. Compared to 1% amylase , the clones of α-amylase showed a steeper increase in the diameter of clear zone from , thus we can conclude that the effect of α-amylase on starch is proportional to the time taken.
Discussion
α-amylase is preferred in our project as it continuously produces α-amylase to degrade starch. Following the trend in the graph, it can degrade more starch in the long run. As the process of anaerobic digestion lasts for a long period, continuous increase in degrading effect fits better to our aim.