Gene Expression of Promoter Lacy

...

Ligation

The purpose of this section of the experiment was to compile sticky ends of the promoter and the vector to be base-paired together, fused by DNA ligase and create a recombinant circular DNA. The process was begun by first thawing the T4 ligase mixture. Two ligation reactions were set up in 200 μL tubes: the first one was the ligation reaction and the second was the vector only control. Two μL of the ligase mixture, 10 μL of vector DNA, and 8 μL of inserted DNA were placed in the ligation tube contained The vector tube contained 8 μL of water, 2 μL of the ligase mixture and 10 μL of vector DNA. The contents were mixed by pipetting up and down and were then left to sit for 10 minutes.

Transformation

For the transformation experiment four tubes each containing 50 μL of cells were set up. Next, 2 μL of ligation reaction was added to tube 1, 2 μL of the vector only reaction was added to tube 2, 2 μL of pMS201-crp as a positive control was added to tube 3 and no DNA was added to tube four to serve as a negative control. The tubes were then kept on ice for approximately 15 minutes then went through a heat shock when being placed in a 42 degree Celsius hot water bath for 45 seconds. Following this stem 250 μL of SOC media was added to each tube and the tubes were mixed. The tubes were then placed in a 37 degree Celsius shaker for 45 minutes. After this time, 200 μL of each sample was plated out onto the center of four separate kanamycin plates. These plates were then placed in a 37 degree Celcius incubator and left to grow for one week.

Isolation of Recombinant DNA

In this step of the experiment recombinant DNA was isolated using a Miniprep kit. A liquid culture was obtained and 1.5 ml of the cell culture was placed into a 1.5 ml tube and spun down in a centrifuge at 8000 rpm for 60 seconds. Supernatant was removed from the tubes without disturbing the cellular pellet present at the bottom of the tube. The cellular pellets were then suspended in 250 μL of Buffer P1, 250 μL of Buffer P2, and 350 μL of Buffer N3. The tubes were mixed periodically between adding the buffers to avoid cell clumps. The tubes were then centrifuged for 10 minutes at max rpm to form a condensed pellet of chromosomal DNA, denatured proteins, cellular debris and SDS. After decanting the supernatant, the QIAprep spin column was centrifuged for 60 seconds and then washed by adding 500 μL of buffer PB, and then centrifuged for 60 seconds again. The spin column was then washed again by adding 750 μL of buffer PE and centrifuged for 2 minutes. The QIAprep spin column was then placed in a clean 1.5 mL tube along with 50 μL of Buffer EB and centrifuged for 1 minute. The recombinant DNA was then present in the micro centrifuge tube and ready to be used for the PCR reaction.

PCR

For the PCR reaction to occur 3 sets of primers were obtained to set up PCR tubes in accordance to predetermined conditions as seen in table 1 which can be seen below. All tubes were kept on ice and the PCR mix was added last. Once the solutions are added to each tube, the thermocycler settings were changed to those for this specific experiment.

Tubes

H20

Recombinant DNA

lacY

araB

mglB

PCR mix

Total Volume

Control

22.5 ul

-

2.5 ul

-

-

25 ul

50 ul

lacY PCR

18.5 ul

4 ul

2.5 ul

-

-

25 ul

50 ul

araB PCR

18.5 ul

4 ul

-

2.5 ul

-

25 ul

50 ul

mglB PCR

18.5 ul

4 ul

-

-

2.5 ul

25 ul

50 ul

Table 1- Chart depicting the perpetration for PCR reaction. Various primers were used in order to uncover the unknown one used in the experiment.

Analyze PCR Product with Agarose Gel

To analyze the PCR product, the Agarose gel was first prepared using the same technique previously described. Twenty microliters of each PCR product and 5 μL of loading dye were added into 5 tubes. Each solution was mixed and then inserted into lanes 1-5 on the gel in the order of DNA Ladder, Control PCR, lacY PCR, araB PCR, and mglB PCR. The gel was then run for 45 minutes at 120 Volts to separate the DNA into segments, then analyzed using a UV box.

Time Series Experiment

In order to figure out if the promoter used in this experiment can be turned on by other inducers and whether it will be turned off by the presence of glucose, five experiments were set up in five separate 1.5 mL tubes. Experiment 1 contained the original plasmid pMS201-crp plus 20 μL of the lacY inducer. Experiment 2 contained the recombinant DNA plus 20 μL of Lactose. Experiment 3 contained the recombinant DNA plus 20 μL of L-arabinose. Experiment 4 contained the recombinant DNA plus 20 μL of Casamino acids. Experiment 5 contained the recombinant DNA, 20 μL of the lacY inducer and 20 μL of Glucose. Additionally, 1 mL of minimum media was added to each experiment solution. After mixing, 220 μL of each mixture was pipetted into wells A1 –