Team:Pitt/Skin Probiotic/Dehydrogenase/Methods

Methods for Desaturase

1. Double restriction digestion with the restriction enzymes EcoRI, SpeI, XbaI, or PstI
   We prepared the double restriction digestion according to table 1. After thoroughly yet gently pipetting the mixture up and down several times, the reaction was then incubated at 37 ℃ for 30 minutes. After incubated, the reaction was immediately loaded onto a pre-made 0.8% agarose gel.
   Table 1: Reagent Table for Double Restriction Digestion.
   

   Components	Amount (ul)
   Sterilized Double Distilled Water (SDDW)	13
   Fast Digest Buffer	2
   Plasmid DNA	7
   Enzyme 1	1
   Enzyme 2	1
   Total	23

2. Gel Electrophoresis
   This technique was used to visual or separate sample with different size DNA fragments. Most often gel electrophoresis was used to perform both tasks at the same time.
   The 0.8% gel was prepared by using mixing 0.24 g of low point melting point agarose in 30 mL of 1x TBE. The mixture was heated until all visible chunks of agarose gel melted. While waiting for agarose gel to cool, 1 uL of Ethidium Bromide was added to the solution and mixed thoroughly.
   Pour the solution into a gel caster with combs of desired number of wells. Typically 1 – 2 uL of DNA ladder (1kb or 1kb plus) and 5 uL of each digestion reactions were loaded into the wells. All gel electrophoresis were run at 100 volts for 30 – 60 minutes.
3. Gel Purification
   This technique was used to separate desire DNA fragment from an agarose gel sample. The samples were first separated on an agarose gel and then the fragment of interest will be cut out of the agarose gel by using a UV light box and razor blade. In order to purify the DNA from the agarose gel, the Wizard® SV Gel and PCR Clean-Up System was used for the purposes of purification.
4. Sticky End Ligation
   This technique was used to construct the new parts by ligating parts that have Biobrick standard sticky ends. A mixture of reagents was prepared according to Table 2 and mixed thoroughly by pipetting the solution gently up and down. After mixing, the reaction mixture was incubated at room temperature for 30 – 60 minutes. The mixture was then incubated at 65 ℃ for 10 minutes for heat inactivation of T4 ligase. Transformation reaction typically followed immediately while the ligation reaction was chilled on ice.
   Table 2: Sticky End Ligation Reaction Reagent Table
   

   Components	Amount (ul)
   T4 Ligase	1
   Ligase Buffer	1.5
   Vector Backbone	3.5
   Insert	9
   SDDW	5
   Total	20

5. Transformation in E. coli Chemical Competent Cells
   This technique was used to maintain as well as to harvest more of the plasmid we created by the ligation reactions.
   Chemically competent E. coli cell culture was thawed on ice for approximately 10 – 20 minutes. Once the cell culture thawed, 50 uL of cell culture was transferred into new microcentrifuge tube. The cell culture was kept on ice before and after transferring to new tube. A total amount of 5 uL of plasmid DNA solution was pipetted into 50 uL of cell culture—this mixture was subsequently flicked 6 times to ensure thorough mixing without agitating the cells. The reaction was chilled on ice for 2 minutes and then heat shocked at 42 ℃ in either a heat block or water bath for 45 seconds. After heat shock the reaction was chilled on ice again for 2 minutes and then was added to 950 uL of liquid LB or SOC media. The cells were then allowed a recovery time of 60 minutes prior to being plated onto antibiotic containing LB plate.
6. Isolating Genomic DNA with Qiagen mini-prep kit
   This was used to harvest plasmid from E. coli cells. Prior to experiment, 3 uL of antibiotic was added to 3ml of liquid LB media to obtain a dilution of 1:1000. Single colonies were grown in the antibiotic infused LB media for 14-16 hours. The resulting cell culture was treated with Qiagen mini-prep kit in accordance to the Qiagen mini-prep kit protocol.

Experimental Design for Constructing Desaturase Part:

Figure 2: A New System of Δ12 Desaturase Part Infused with mRFP1 Fluorescence Protein

1. Cut mRFP1 with EcoRI and XbaI to obtain the vector back bone.
2. Run agarose gel to isolate vector backbone fragment.
3. Gel purification.
4. Cut Δ12 desaturase (BBa_K1027002) with EcoRI and SpeI.
5. Run agarose gel to isolate desaturase part.
6. Ligate the desaturase part into the vector backbone containing mRFP1.
7. Transformed the plasmid into E. coli competent cells.
8. Spread on LB + CAM plate and incubate 14-16 hours at 37 ˚C.
9. Pick single colony to grow in LB + CAM culture.
10. Mini-prep the cell culture.
11. Perform diagnostic digest with XbaI and PstI and isolate mRFP1–desaturase insert.
12. Run agarose gel isolate the part.
13. Gel purification.
14. Transform into Hsp60 with RBS vector backbone cut with SpeI and PstI.

Timeline

Next Page

Previous Page