Final Project - Cloning TRK1/pSMFP2-C1

During fermentation, the ethanol produced can become too concentrated and kill the yeast. However, some yeast strains are more resilient to higher ethanol concentrations which allows for greater production for brewing or ethanol for fuels. One of the genes associated with this resilience is TRK1–a potassium importer. TRK1 helps to create an ion gradient to allow removal of hydrogen ions from the cell which in turn allows yeast to live longer in acidic conditions. The goal of the experiment is to determine whether certain environmental conditions up-regulate TRK1 expression. To quantify expression, TRK1 must be fluorescently labeled in a fusion protein with a fluorescent tag to eventually clone the plasmid into yeast.

Isolate the TRK1 coding gene

Reverse transcribe TRK1 into cDNA

Select primers and enzyme restriction sites for the TRK1 gene and via PCR, amplify TRK1 cDNA.

Digest and ligate TRK1 gene into a fluorescently labeled fusion plasmid

Run a restriction mapping test to determine effectiveness of gene insert. i.e. whether TRK1 was fused correctly with the plasmid

1 mol = (323 g/bp) * (3708 bp) = 1.20*10^6 g/mol

(10^7 molecules/6.02*10^23 molecules) * (1.20 * 10^6 g/mol) = 0.020 ng

0.020 ng * (µL/600ng) = 3.33*10^-5 µL

To have this volume capable for pipetting, make a 100000-fold dilution and pipette 3.33 µL

600 ng/µL diluted 10^5-fold is 0.0060 ng/µL

0.020 ng / (0.0060 ng/µL) = 3.33 µL into PCR for 10^7 copies

6X * Y = 1X(25+Y)

Y = 5

50X*V1 = 500mL * 1X

V1 = 10mL

0.02mg/mL * 50mL = 1g

15mg/mLX = 5*10^-4 mg (50+ X)

X = 1.67 uL

5V for every cm = 100V

Negative Control: To test for contamination, do not add cDNA and add water instead

Positive Control: To test for working primers, add cDNA of yeast genome

Technical Control: To test for reverse transcription accuracy, add beta actin gene

Minimum volume: (3.6 + 3.6 / 2) / 5% = 72 uL

Minimum volume: 18 uL

Vector Concentration: 0.6 absorbance units after a 1:19 dilution

0.6 * 50ng/uL * 20 = 600 ng/uL

Insert Concentration: 1.6 absorbance units after a 1:9 dilution

1.6 * 50ng/uL * 10 = 800 ng/uL

y = 2.35

3.35x = 250 ng

x = 74.6 ng vector

175.4 ng insert

1. Refer to the protocol (Reverse Transcription From RNA) attached to this entry for detailed instructions on how to prepare TRK1 RNA conversion to cDNA for PCR.

2. Design primers to amplify TRK1 CDS

a. Find restriction sites for the MCS of the vector that are not present in the TRK1 gene

4. Create primer pair to anneal to TRK1

a. Keep in mind melting temperatures (no more than 5 degrees different) and GC content (around the same).

5. Add the restriction site as a 6 bp overhang

6. Calculate the volume for stock solutions for 1 reaction and a master mix for 6 reactions–includes an additional fudge factor.

7. After determining the respective volumes, add the reagents into a master mix tube, excluding cDNA.

a. The thermocycler should be warming up to 94 C at this point and the preparation should be taking place on ice

8. Separate the master mix into 5 different reaction tubes

a. Negative control contains water instead of cDNA

b. Positive control contains yeast cDNA

c. Technical control is the reverse transcription of the beta actin gene to ensure that all of the TRK1 was properly reverse transcribed

9. Run the PCR for 35 cycles using the conditions below

10. Run the gel

11. After verifying the PCR and reverse transcription was successful, we can conduct a digest on the amplicon and plasmid

12. Using 3:1 amplicon to vector ratio, we run a ligation

a. Assure that the gene of interest is in frame with the fluorescent tag and has the correct orientation

13. Run a restriction mapping digest using enzymes BmgBI and KasI

14. Run a gel for the mapping digest

a. Positive control: Insert cut at the BmgBI restriction site

b. Negative control: No enzymes

15. Farm recombinant in E. coli (not done in benchling)

​Inaccessible DNA Sequence

​Inaccessible DNA Sequence (Plasmid)

​Inaccessible DNA Sequence (Final Recombinant Plasmid)

Minimum volume: (5.4 + 2.7 / 2) / 5% = 81 uL

As expected, the recombinant plasmid (negative control) was ~8300 bp and the positive control was successful which shows the cut site was present.