Test

Cycle #1 - Gene Fragment Cycle

Experimental design

After extracting Lumbrokinase tissue, we cleansed the RNA sample by pipetting and adding isopropanol, allowing the RNA to precipitate and removing the transparent layer on top of the RNA to perform measurements of RNA concentration and quantity. We produced agarose gel and loaded it in the RNA to determine whether RNA was degraded or contaminated by chromosomal DNA.

Procedures

• Add 20 µL of DEPC Water to extracted RNA sample, then mix well using the micropipette
• Use nanodrop to measure the RNA concentration and quality
• Produce 1% agarose gel
• Extract 100 ng RNA and transfer to a new microcentrifuge tube, add an equal mass of 2X RNA loading die, mix well
• Slowly remove the comb, then transfer the produced agarose gel into the gel tray. Ensure the negative electrode is on top and corresponds with the black pole, then add an appropriate volume of 1x TAE buffer liquid so that the gel is submerged
• Carefully add the mixed RNA sample into the wells using the micropipette and prevent contact with the well to avoid losing content
• Connect the electrodes, then switch the power supply adjustment to voltage mode, set to 110 V for 15 minutes, then observe the results using the gel image system.
• Extract the RNA and determine successful extraction of the Lumbrokinase gene

Results

RNA extraction was performed on four different earthworms, with three of them having similar diets and dehydration while one was treated normally, and the results indicate the samples with the highest purity of total RNA.

Nanodrop (table 1)

Table 1 indicates the concentration of the three samples with altered diet and hydration to be from 375 ng/uL to 560 ng/uL, while the 260/280 and 260/330 values of nucleic acid purity still indicate certain impurities in the extracted RNA.

Our team also performed gel electrophoresis to confirm the quality of the extracted total RNA by running the four samples, in which the results reveal multiple bands for sample 1, 2, and 3, while sample 2-2 only has one visible band. Only having one visible band could mean multiple things, but some explanations include the RNA sample being degraded or contaminated by other molecules, indicating the 2-2 sample is not suitable for future experimentation.

Although the nanodrop results indicate slight impurities, we improved the concentration results significantly from when we did not apply methods of fasting, dehydration, and homogenization of tissue.

Nanodrop (table 2)

Table 2 indicates a varying concentration of 280 ng/uL to 810 ng/uL, which is a larger deviation compared to after the change in diet and extraction techniques in table 1. Further, the nucleic acid purity is also significantly lower on average compared to table 1. Thus, our team improved the RNA quality by implementing our gene fragment cycle, resulting in a more balanced and higher concentration, as well as nucleic acid purity.

Cycle #2 - Plasmid Cycle
(pGEM-T EASY vector → pET-22b)

Experimental design

We chose to conduct blue-white screening to confirm the successful transformation and insertion of the lumbrokinase gene with pGEM-T EASY vector into E.coli DH5-alpha. We looked at the presence of white and blue colonies to determine success. For the transformation process, we first heated the cell membrane of E.coli DH5-alpha before introducing the plasmid into small holes in order to let it fuse.

Apparatus

• Pipetman (P1000, P200, P20, P10, P2.5)
• Ice bucket with ice
• Dry bath (set at 42oC)
• Glass Bead Sterilizer (# FYO001-100G, Yeastern Biotech Co., Ltd)
• Incubator (37oC)

• E.coli DH5α (# FYE678-10VL, Yeastern Biotech Co., Ltd)
• Amp LB agar plate (J63197.EQF, Thermo Fisher)
• X-gal (20 mg/mL) (R0941, Thermo Fisher)
• IPTG (100 mM) (R1171, Thermo Fisher)

• Defrozen E.coli DH5α on ice
• Use a micropipette to add ligation reaction solution and mix it well, then place it on ice for 15 minutes
• Move the bacterium solution to a 42oC dry bath for 45 seconds to react, then place it on ice for 15 minutes
• Pour appropriate amounts of glass bead into Amp LB agar plate and use a micropipette to add 50 µL of X-gal and 100 µL of IPTG. Shake the mixture well at a regular speed until there’s no liquid on its surface, then pour the glass bead out and move the agar plate to a 37oC incubator for drying
• Pour appropriate amount of glass bead into X-gal/ IPTG/ Amp LB agar plate, mix bacterium solution well with a micropipette then add it to agar plate. Shake the bacterium well at a regular speed until there’s no liquid on its surface, then pour the glass bead out and move the culture medium to a 37oC incubator. Cultivate it for 14-16 hours

Results

0

The white colonies indicate a functional β-galactosidase enzyme was not formed, which means that there was no foreign DNA inserted into the lacZ gene. Our experiment wanted the blue colonies as validation since our lumbrokinase is a foreign DNA inserted into the lacZ gene, which disrupts β-galactosidase enzymes from forming, thus resulting in a blue colony.

Cycle #3 - Primer Design Cycle

Experimental design

After insertion of lumbrokinase into the pFadH_Lumbrokinase_pET-22b, the primers we had designed as well, as the replaced lac operator for the FadR regulator, was confirmed by various tests that included gel electrophoresis of the annealed bands cleaved at three parts of the vector (more information on our proof of concepts page). We performed two gel electrophoresis that included primer annealing of four primer samples with different treatment and a large scale restriction enzymatic reaction – to confirm the removal of the lac operator from pGEM-T EASY vector when transformed into the pFadH_Lumbrokinase_pET-22b.

Primer annealing Apparatus

• Pipetman (P1000、P200、P20、P10、P2.5)
• Vortex
• PCR machine
• PCR tube
• Electrophoresis tank and module
• UVP imaging system

Primer annealing Materials

• Primer
  

  a. T7_FadR_forward:

  gatcTAATACGACTCACTATAGGCGACGGCTAAATTAGAACTCATCCGACCACAT

  b. T7_FadR_reverse:

  gatcTAATACGACTCACTATAGGCGACGGCTAAATTAGAACTCATCCGACCACAT

• 10X annealing buffer (1 M K-acetate, 0.3 M HEPES-KOH, 20 mM Mg-acetate, pH7.4)
• ddH2O
• Agarose
• Tris-acetate-EDTA (TAE) buffer
• DNA ladder

Primer annealing Procedures

• Prepare annealing mixture as the following table

  Components	1X
  T7_FadR_forward (50 μM)	9 uL
  T7_FadR_reverse (50 μM)	9 uL
  10X annealing buffer	2 uL
  Total volume	20 uL

• Use PCR machine anneal primers:

  a. 95oC, 78oC, 74oC, 70oC, 67oC, 63oC, 60oC, 56oC, 53oC, 50oC, 48oC, 46oC, 44oC, 42oC, 40oC, 39oC, 37oC, 36oC, 35oC, 34oC, 33oC, 32oC, 31oC — 5 min in each step

  b. 30oC, 28oC, 26oC, 24oC, 22oC, 20oC — 10 min in each step Hold at 4oC

• Dilute 50X with ddH2O
• Heat 20 uL annealing product at 95oC for 10 min as negative control
• Run 3% agarose electrophoresis

Primer annealing Results

Figure 12 indicates that the four primer samples were properly annealed, meaning the annealed bands moved slightly faster than the annealed and heated band, the forward band, and the reverse band. Thus, our team concluded that it was the structure of the bands that caused this difference since annealed bands have nitrogenous bases concealed internally (positive charge), meaning it would be more negatively charged compared to the other bands.

Large scale restriction enzymatic reaction Apparatus

• Pipetman (P1000、P200、P20、P10、P2.5)
• Incubator (37oC)
• Electrophoresis tank and module
• UVP imaging system

Large scale restriction enzymatic reaction Materials

• Lumbrokinase-pET-22b (+) plasmids (526.44 ng/uL)
• XbaI
• BglII
• 10X NEBuffer 3.1
• Agarose
• Tris-acetate-EDTA (TAE) buffer
• DNA ladder

Large scale restriction enzymatic reaction Procedures

Double-digest the plasmids by XbaI and BglII

1. Run 1% agarose electrophoresis

Large scale restriction enzymatic reaction Results

Figure 13 includes the gel study of three samples of the pGEM-T EASY vector for confirmation of the removal of the lac operator from the pGEM-T EASY vector. The three vectors of uncut, cleaved by Bglll and Xbal, and only cleaved by Bglll indicate that the missing band from the both cleaved vectors is the lac operator.

Cycle #4 - BL21 and Protein Expression Cycle

Experimental design

We needed to find the optimal parameters for the induction processes of IPTG and oleate, so we chose to use SDS-page and CBR staining for IPTG induction and then SDS-page and Western blotting for oleate induction; ultimately, allowing us to analyze the protein expression quality and compare the results of the two expression systems under different conditions to find optimal production parameters.

IPTG induction

SDS-page and CBR staining Apparatus

• Rotary shaker
• SDS-PAGE module
• Plastic box
• 100oC incubator

SDS-page and CBR staining Materials

• 30% acrylamide mix
• ddH2O
• 1.5 M Tris-HCl (pH 8.8)
• 0.5 M Tris-HCl (pH 6.8)
• 10% SDS
• 10% ammonium persulfate
• TEMED
• Tris-glycine SDS running buffer
• Coomassie brilliant blue

SDS-page and CBR staining Procedure

• Add 5X sample buffer and heat at 100oC for 5 min
• Run a 15% SDS-PAGE
• Stain with CBR

SDS-page and CBR staining Results

Figure 14 indicates that the IPTG induction has been effective since around the twenty-four mark there is a visible band that is approximately at 34.23 kDa of lumbrokinase (shown by the red arrow).

SDS-page and Western blotting Apparatus

• Rotary shaker
• SDS-PAGE module
• Plastic box
• PVDF membrane
• Transfer system
• Refrigerator (4°C)
• UVP imaging system

SDS-page and Western blotting Materials

• 30% acrylamide mix
• ddH2O
• 1.5 M Tris-HCl (pH 8.8)
• 0.5 M Tris-HCl (pH 6.8)
• 10% SDS
• 10% ammonium persulfate (APS)
• TEMED
• Tris-glycine SDS running buffer
• Antibodies
• 1% bovine serum albumin (BSA)
• PBST
• Methanol
• Transfer buffer

SDS-page and Western blotting Procedure

• Add 5X sample buffer and heat at 100°C for 5 min
• Run a 15% SDS-PAGE
• Transfer to PVDF membrane
• Block by 1% BSA
• Incubate with first antibody
• Incubate with secondary antibody
• Take UVP images

SDS-page and Western blotting Results

Figure 15’s results show an evident supernatant around twenty-four hours that is close to the approximate 34.23 kDa of lumbrokinase, so we speculated that the additional band meant production of lumbrokinase was reaching its peak at the twenty four hours.

O’Leate induction

SDS-page and CBR staining Apparatus

• Rotary shaker
• SDS-PAGE module
• Plastic box
• 100°C incubator

SDS-page and CBR staining Materials

• 30% acrylamide mix
• ddH2O
• 1.5 M Tris-HCl (pH 8.8)
• 0.5 M Tris-HCl (pH 6.8)
• 10% SDS
• 10% ammonium persulfate
• TEMED
• Tris-glycine SDS running buffer
• Coomassie brilliant blue

SDS-page and CBR staining Procedure

• Add 5X sample buffer and heat at 100°C for 5 min
• Run a 10% SDS-PAGE
• Stain with CBR

SDS-page and CBR staining Results

Figure 16 and 17 show how the optimal parameters of oleate induction are first visible at 2.5 micromolars, but they remain relatively equal as time passes (more information in our results page).

SDS-page and Western blotting Apparatus

• Rotary shaker
• SDS-PAGE module
• Plastic box
• PVDF membrane
• Transfer system
• Refrigerator (4°C)
• UVP imaging system

SDS-page and Western blotting Materials

• 30% acrylamide mix
• ddH2O
• 1.5 M Tris-HCl (pH 8.8)
• 0.5 M Tris-HCl (pH 6.8)
• 10% SDS
• 10% ammonium persulfate (APS)
• TEMED
• Tris-glycine SDS running buffer
• Antibodies
• 1% bovine serum albumin (BSA)
• PBST
• Methanol
• Transfer buffer

SDS-page and Western blotting Procedure

• Add 5X sample buffer and heat at 100°C for 5 min
• Run a 10% SDS-PAGE
• Transfer to PVDF membrane
• Block with 1% BSA
• Incubate with first antibody
• Incubate with secondary antibody
• Take UVP image

SDS-PAGE & Western blotting Results

Figure 18 and 19 exhibit the samples under varying concentrations of oleate induction. After analysis, we determined the least amount of time and concentration for lumbrokinase to produce optimally is at 2.5 micromolars oleate concentration after just six hours.