Enzyme Activity Experiment Report (chitinase, protease)
The main purpose of this experiment is to measure the activity of two target enzymes, chitinase and protease, in a TOP 10 expression system. The sequences of both chitinase and protease were cloned from a entomogenous fungus called verticillium lecanii. Testing the efficiency and the effects of these enzymes’ expression is a fundamental portion of the whole project. We hypothesized that the enzyme activity (defined by active unit divided by bacteria population) is positively related to a given period of time. We obtained factors from bacteria density in culture to the degradation amount of substrate via visible spectrophotometer.
Seq 1: K2224001
Promoter+RBS+Lecanicillium lecanii strain 432 endochitinase (chit1)+Terminator
Seq 2: K2224002
Promoter+RBS+Lecanicillium lecanii strain CA-12 cuticle-degrading proteinase (Pr1)+Terminator
Enzyme Activity Definition:
The enzyme activity is defined by the amount of N- acetyl glucosamine that produced by Chitinase' s hydrolysis to chitin divided by the number of cells (or bacteria concentration). Since the product, N- acetyl glucosamine, can lead to light absorbtion peak at 585nm and the light absorption can be detected by Spectrophotometer, we can figure out the relative activity of enzyme DQ412944.
Considered all the properties of E. coli expression system, there are serval things that can influence the expression of a particular enzyme. To understand and categorize the factors, the expression process of enzyme (protein, specifically) need to be broke down into three stages, which are target plasmid replication, transcription and translation (include further process to structure protein). Among these stages, transcription and translation affects the expression efficiency mainly.
In this experiment, the influence to chitinase and protease expression in TOP10 expression system is mainly measured in two factors, which is bacteria population and enzyme activity. To be specific, bacteria population determines the size of bacteria population that is capable for enzyme expression. Enzyme activity is the ability or effectiveness of catalyzing reactions.
Considered that the condition in natural environment will be much more complicate and unpredictable, designing appropriate experiments and dividing factors correctly are especially vital.
More importantly, our experiment is constructed by learning previous paper carrying out resemble experiments. According to our studies, previous experiments mainly focused on characterizing the enzyme (Chitinase and Protease), so the researchers expressed the gene (in mainly two different ways, culture the verticillium lecanii then extract the enzymes, and modified the sequences and express it in E. coli expression systems) and tested the enzyme activity in different temperature and different pH. One study tested the enzyme activity to figure out the enzymic mechanism that helped verticillium lecanii invade host coccid. Our experiment aims to explore the possibility in developing this mechanism into a product. Therefore, we designed our experiments by relating the enzyme activity to culturing time, which enables us to acquire the optimum culturing time when using the product.
According to the bibliographical material, the re-expression of chitinase is completed in JM109 (a type of E. coli strain) expression system with plasmid backbone pMD18-T. The re-expression of protease is completed in BL21(DE3) with plasmid backbone pMD18-T. Both of the template experiment in the material use Amp as an antibiotic. In our experiment, we take TOP10 as the expression system, using standardized iGEM plasmid backbone pSB1C3 and Chl as antibiotic.
According to the analysis above, we predict that the enzyme activity is positively related to time within a period.
1. Materials and Methods for Chitinase Activity Detection
a) TOP10 of Escherichia coli and LB solid medium materials (yeast extract, tryptone, sodium chloride, agar powder, DD water) LB liquid culture medium material (yeast extract, tryptone, sodium chloride, DD water)
b) inoculation ring, antibiotic (chloramphenicol), Petri dish, adjustable transfer gun, constant temperature shaking incubator, LB medium, coating rod, alcohol lamp, EP tube
c) balance, water bath, centrifuge, visible spectrophotometer, 1 mL glass cuvette, distilled water
d) chitinase activity detection kit:
Extract: liquid 100mL x 1 bottles, stored at 4℃.
Reagent 1: liquid 20mL x 1 bottles, stored at 4℃.
Reagent 2: liquid 10mL * 1 bottles, stored at 4℃.
Reagent 3: liquid 10mL * 1 bottles, stored at 4℃.
Reagent 4: liquid 20mL * 1 bottles, stored at 4℃ away from light.
a) Draw Standard Calibration Curve for Bacterial Concentration: prepare LB medium.
Take the available preserved TOP10 strain, pick the bacteria strain by using a platinum ring beside a fire and then draw lines on solid LB medium. Culture the inoculated medium in constant temperature incubator for 24h, temperature at 37 ℃ ±1 ℃ in incubator. Pick monoclonal bacteria colonies, inject in 20mL liquid LB medium, shake the medium in constant temperature shaking incubator (110r/ min, 3cm oscillation amplitude) for 24h.
Take 0. 4mL cultured bacteria fluid in logarithmic growth phase, cultured in 20mL nutrient bacteria medium, then shake the medium for 4h to get bacteria fluid in stable phase.
Dilute the cultured bacteria fluid in stable phase with liquid LB medium (for 5, 10, 20, 40 times). In 660nm standard wavelength, measure the absorbance (ABS) of this group of samples different bacteria concentration. (Zeroing before testing with 1x liquid LB medium).
Determine the colony numbers of four different dilution times in turns in a sterile room next to the flame. Each sample was formulated in several concentration gradients, and 1 mL of each sample was injected into the dish with sterilized pipette. Inject about 15 mL ,45～50 ℃ solid LB medium, then turn the dish so that the sample with the medium can be fully mixed. When medium solidified, turn the plate. Place the medium in 37 ℃±1 ℃ incubator to culture for 24h, count the number of colonies. With eye observation, the colony number was counted and recorded. The average colony number of each dilution plate can then be obtained.
b) Glycerol Bacteria Recovery: Use inoculation ring to dip into glycerol bacteria and then draw line on target solid LB culture medium, or use glycerol bacteria to coat on solid LB culture medium directly for recovery, pick few positive bacteria culture colonies for shaking.
c) Crude Enzyme Extraction: according to the ratio between the number of cells (104): the volume of extraction solution (mL) as 500~1000 : 1 (recommendation 5 million cells with 1mL extract), ultrasonic cell crushing ice bath (ultrasonic power 300W, 3S, the total time interval of 7 s, 3min); centrifuge 20min at 4℃ with rotating speed of 12000rpm. Place on the ice for later inspection.
2. Materials and Methods for Protease Activity Detection
a) visible spectrophotometer, water bath pot, magnetic stirrer, adjustable transfer gun, 1mL glass cuvette, 1.5 mL EP tube and distilled water. Chitinase activity detection kit:
b) reagent 1: liquid, stored at 4℃
Reagent 2: powder, stored at 4℃. Dissolve with 10 mL distilled water before use.
Reagent 3: powder, store at 4℃ away from light. Before adding 10 mL reagent, boiling water dissolves.
Reagent 4: powder, stored at 4 degrees. Dissolve with 50 mL distilled water before use.
Reagent 5: liquid, stored at 4 ℃.
Standard: liquid, 0.25 mol/mL standard tyrosine solution, stored at 4 c..
c) inoculation ring, antibiotic (chloramphenicol), Petri dish, adjustable transfer gun, constant temperature shaking incubator, LB medium, coating rod, alcohol lamp, EP tube
a) Draw Standard Calibration Curve for Bacterial Concentration.
b) Glycerol Bacteria Recovery.
c) Crude Enzyme Extraction: according to the number of cells (104): reagent volume (mL) ratio of 500~1000:1 (5 million cells with 1mL reagent), ultrasonic cell crushing ice bath (ultrasonic power 300W, 3 seconds, 7 seconds between the total time, 3min and 8000g); 4, C, centrifugal 10min, Pick supernatant and place on ice to for further measuring. (Note: get the number of bacteria by measuring the ABS value.)
1. Standard Calibration Curve for Bacterial Concentration:
2. Chitinase Activity Measurement
From the first set of data, we can clearly discover the linear relationship between sample bacteria concentration and the absorbance. Data suggest that as the concentration of bacteria fluid becomes greater, the relative absorbance increase proportionately. This result justified our assumption.
From the second set of data we can reach an agreement that our TOP10 Expression System is suitable for the expression of plasmid K2224001, which CDS is the modified chitinase. However, the chitinase activity curve with insufficient data only shows an uncertain relationship with time. We can define this relationship as a linear relationship but since it is weak and uncertain, further research and experiment should be taken, until a confirmation can be done.
Unfortunately, the expected protease activity measurement is not successfully done since the measurement kit is invalid by inappropriate preservation operation.