Molecular Cloning

Polymerase Chain Reaction (PCR)

FastPfu and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase.

1. The master mix for reactions with FastPfu DNA polymerase contained:

2. The master mix for reactions with Phusion DNA polymerase contained:

3. The master mix for reactions with Taq DNA polymerase contained:

4. All temperature profiles were optimized according to manufacturer’s protocol, he melting temperature of primers, and the length of the desired PCR products.

Basic temperature profiles

Fragment DNA isolation from agarose gel

Agarose Electrophoresis

A mixture of various sized DNA fragments were separated in an agarose gel (from 0.8 to 1.5% agarose in 1x TAE buffer ethidium bromide) at a constant voltage of 150 V.

UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide.

Fragment Isolation from agarose gel

The band with the desired DNA fragments were excised from the gel, using a clean scalpel.

DNA was isolated from the gel slice with Gel Extraction Kit according to the manufacturer’s protocol.

Purity and amount of DNA was determined using NanoDrop.

Restriction digest

1. To digest the desired DNA restriction reactions were prepared as follows:
For analysis of cloned DNA
2µl of the appropriate restriction buffer (10X)
0.5 µL restriction enzyme
Bring volume to 20 µL with nuclease-free water.


Or

For isolation of specific DNA
2µl of the appropriate restriction buffer (10X)
Up to 2 µL restriction enzyme
Bring volume to 50 µL with nuclease-free water.

2. The sample was incubated at optimal temperature for the restriction enzymes.
3. Analysis of fragmented DNA was done by gel electrophoresis.
4. Desired DNA fragment was excised and purified using suitable DNA purification kit.

Ligation

T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA.

Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA).

A ligation mixture was prepared:

1X ligase buffer (10X)
1 µL T4 ligase (3 U/µL)
Bring volume to 10 µL with nuclease-free water.

Reactions were incubated at 17 °C for 4 to 18 hours.

After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria).

Culturing bacteria

For plasmid DNA propagation two bacterial strains were used: DH5alpha and TransT1.

Growth media for bacteria

Luria Broth (LB) : 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.

LB agar plates: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.

Transformation of bacteria

E. coli DH5alpha and TransT1 competent cells were used for the propagation of plasmid DNA.

100 µL of competent cells were thawed on ice.
50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution).
A mixture of cells and DNA solution was incubated on ice for 30-60 minutes.
The mixture was heat-shocked for 3 minutes at 42 °C.
Cooled for 3 minutes on ice.
500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance.
The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.

Plasmid DNA isolation

MINI PREPs for analysis and sequencing

A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol).
Bacteria were grown over night at 37 °C with agitation.
Plasmid DNA was isolated from 6-10 mL of over-night culture with plasmid miniprep kit according to the manufacturer's protocol.
Amounts ranging from 6-10 µg of plasmid DNA were obtained.
The purity and concentration of the isolated DNA was analyzed using NanoDrop.

Yeast Transformation Protocol

1. Inoculate a single colony of the desired yeast strain to be transformed into 5 ml YPD or selective media and grow overnight at the appropriate temperature.
2. From the above overnight culture, inoculate into 5 ml YPD/selective media the appropriate amount to make the culture's optical density at 600 nm (OD600) equal to 0.1.
3. Shake at the appropriate temperature until OD600 is between 0.6 and 1.0 (Normally I won’t let the OD reach over 1.0. Depends on strains, it may take 6-8 hours).
4. Pellet cells 5 min at 3,000 rpm at room temperature.
5. Wash the cells with 1ml ddH2O and transfer them into 1.5ml eppendorf tube. Pellet the cells at 3000rpm for 1min and remove the supernatant.
6. Wash the cells with 1 ml 0.1M LioAc/TE buffer. Pellet cells and resuspend in 100ul of 0.1M LioAc/TE.
7. Aliquot 20 ul of cell suspension into each of tube and add 2ul of plasmid DNA
8. Add 80ul of transformation solution and mix well by pipetting up and down for several times

   Transformation Solution (each):

   50% PEG 62.4ul

   1M LioAc 8.22ul

   DMSO 9.58ul

   10mg/ml ssDNA 5ul

9. Incubate the plate at 30°C for 30min (can be longer but not shorter than 30min)
10. Heat shock by placing in a 42°C water bath for 15min
11. Pellet the cells by centrifuging at 3000 rpm for 1min.
12. Remove the supernatant.
13. Wash the cells with 100ul of 5mM CaCl2. Pellet the cells (3000rpm, 1min) and remove the supernatant.
14. Add 100ul of ddH2O, mix well on the plate shaker.
15. Plate cells onto selective plates.

AHL induction and flow cytometry analysis

The response of engineered yeast to AHL quorum sensing signals was examined using pTF4 transformants, where AHL signals would be recognized by LuxR receptors, thereby promoting mCherry fluorescent protein expression. Induced fluorescence was checked using flow cytometry analysis. Samples were prepared as specified below.

1. pTF4 transformant yeast was cultured in SC-His selective medium from 0.1 OD for 8 hours, at 30 ℃, shaking culture.
2. Yeast cells were induced with AHL.

   a. 0.5 μM AHL induction: Added 25 μL AHL (0.1 mM stock solution) into 5 mL pTF4 transformant yeast culture.

   b. Ctrl: Added 25 μL ddH2O as control.

   30℃ shaking culture.

3. Sample fixation: 0.5h, 2h, 8h and 24h post-induction: Collect 500 μM sample and fix in 10% formalin for 15 min. Formalin was removed after fixation and yeast cells were resuspended in 500 μM PBS and stored at 4℃.
4. Samples were subjected to flow cytometry analysis to check for mCherry fluorescence. Negative control was established using native yeast with no mCherry expression.

Western Blot Protocol

1. Sample Preparation

   Add 100µl 0.2M NaOH into 100µl yeast culture media. Incubate at room temperature for 5min. Centrifuge at 7000 rpm for 1min. Remove the supernatant.

   Resuspend the cells in 50µl SDS sample buffer. Incubate at 100℃ for 5min. Centrifuge at 7000 rpm for 1min. Keep the supernatant. Store at -20℃.

2. SDS-PAGE

   The separation gel component is as below:

   Percent（%）	7.5	7.5	10	10	12.5	12.5	15	15
   	2	4	2	4	2	4	2	4
   1.5M Tris-HCl with SDS(PH 8.8) (ml)	2.5	5	2.5	5	2.5	5	2.5	5
   30% Acrylamide(ml)	2.5	5	3.4	6.8	4.2	8.4	5.0	10
   dd H2O (ml)	4.9	9.8	4.1	8.2	3.2	6.4	2.4	4.8
   10% APS (ul)	60	120	60	120	60	120	60	120
   TEMED (ul)	6	12	6	12	6	12	6	12
   Total Volum(ml)	10	20	10	20	10	20	10	20

   stacking gel component is as below:

   	2	4
   1.5M Tris-HCl with SDS(PH 8.8) (ml)	1.25	2.5
   30% Acrylamide(ml)	0.7	1.4
   dd H2O (ml)	3	6
   10% APS (ul)	30	60
   TEMED (ul)	6	12
   Total Volum(ml)	5	10

   Add protein sample to each well.

   Then use 100V to run. When the proteins reach the boundary of separation and stacking gel, you can change the voltage into 140V. Stop when the proteins reach the bottom of gel.

3. Blocking and Antibody Incubation

   Use the 5% milk blocking buffer to block the NC membrane at room temperature for 1h.

   Transfer the NC membrane to first antibody buffer (rabbit anti-flag 1:4000), at 4℃ overnight.

   Use the PBST to wash the NC membrane for 3 times, each for 10min at least.

   Then transfer the NC membrane to the second antibody buffer (rabbit anti-IgG 1:10000). Incubate at room temperature for 1h.

   Use the PBST to wash the NC membrane for 3 times, each for 10min at least.

4. Development

   Add A solution and B solution (1:1), and mix them in the plate.

   Put the membrane into the plate and let the liquid flow past the NC membrane. Take picture of your western result.

Yeast Dry Powder Preparation Protocol

1. Inoculate a single colony of the desired yeast strain into 5 ml YPD media and grow overnight at the appropriate temperature. Determine OD600.
2. Centrifuge at 3000 rpm for 5min. Remove the supernatant. Resuspend the cells in the media left. Transfer them into a 1.5ml eppendorf tube. Centrifuge at 3000 rpm for 1min.
3. Remove the supernatant using pipette. Wash the cells with 120µl ddH2O.
4. Centrifuge at 3000 rpm for 1min. Remove the supernatant.
5. Dry in the vacuum centrifuge at 30℃ for 45min (or more).
6. Grind the clotted yeast. Store in eppendorf tube at room temperature or -20℃.
7. Test after one week.