Team:Saint Joseph/Notebook


Project Notebook

Notebook

day-1 day-2 day-3 day-4 day-5

Day-1 : 07.07.2019

SOC Medium Preparation

20mM glucose is added to the prepared 1L SOB to increase the richness. The SOC medium is not autoclaved so that glucose is not caramelized. The final pH is 6.8-7.

We're moving from LBA to LB Broth. Transition from solid medium to liquid medium. While making this transition, we made a colony selection, placed the colony we have taken on 10ml LB Broth medium and left it for incubation.

Creation of Competent Cells by CaCl2 Method

0.5 ml of overnight E.coli culture is added to 50 ml LB medium and produced at 37 C at 180 rpm until OD600 = 0.3-0.4. The culture is then allowed to stand on ice for 10 minutes. It is then centrifuged at 4 ° C for 14 minutes at a speed of 14,000 rpm. Centrifugation is done to precipitate the cells. 10 ml of 0.1M CaCl 2 was added to the pellet and left on ice for 30 minutes. Centrifuged for 4 minutes at 14,000 rpm at 4 ° C. The pellet is then suspended in 2 ml of 0.1 M CaCl 2, stored at 4 ° C. Finally, 200 microliters of cell suspension is transformed.

E.Coli DH5a Bacteria Breed Transformation:

Added 1-10mL of DNA suspension containing 0.1-1 mg of DNA on 1 to 200 mL of sufficient cell suspension and allowed to stand for 30 minutes on ice. Then it is kept at 42 ° C for 90s and then kept on ice for 2 minutes. 800 mL of LB or SOC medium (37 C) is added and kept at 37 C for 30-90 minutes with gentle agitation. And finally, 100mL / petri LBA selective medium is seeded. Incubated for 1 night. Colony selection is made for the transition from solid medium to liquid medium. We then left the cells to incubate again for 1 day to proliferate in the liquid medium.

Day-2 : 16.07.2019

LB Broth Medium

First we poured 25 gram lb Broth Powder to an autoclaved bottle with screw cap. Then we completed it with 1 liter ddH2O(distilled water). After that, we closed the lid halfly, covered it with foil and glued autoclave tape on it. (The tape which allows us to distinguish). Furthermore, we autoclaved it at 121 degrees for 20 minutes. Lastly, after letting it to cool to room temperature, we kept it at 4 degrees.

LB Agar Medium Making

We prepared 1 liter LB liquid medium in a 2 liter erlenmeyer. Then, we added 15 grams of bacterial agar and we blended it. We autoclaved it at 121 degrees for 15 minutes and cooled it to 50 degrees. We added antibiotics and blended them by spining the bottle strongly. We label the antibiotic from its inside. Lastly we poured the medium into petri plates but while doing it we paid attention that it doesn’t cool down because agar gets polymerised when it cools under 60 degrees. In addition, while adding the antibiotic, we avoided the temperature getting high.

LBA Medium Plantation

We took the medium with a 1- 0,1= 100 microliter pipette and put it in the middle of the petri plates. With the help of an alcohol sterilised and singed baguette we finished the process of propagation.

SOB Preparation

It gets disintegrated in 0,5g NaCl and 20g Tripton 950 ml dH2O. We added 5g Yeast Extract. Yeast Extract is a very rich medium just like a yeast medium. We prepared a proper environment for the bacterias. Then we shaked it until it gets dissolved and we added 10 mL of 250 mM KCL stock. Later, we adjusted the mediums pH to 7 by adding 5g NaOH. We fulfiled the solution with dH2O to 1 liter. We sterilised it by autoclaving 15 to 20 minutes in psi. Before using it, sterilised 2M MgCl2 should be added.( It should be absolutely added after the autoclaving step)

Day-3 : 19.07.2019

Agarose gel electrophoresis

To prepare agarose gel, we mixed 0.5 g of agarose with 75 ml of tae buffer. We heated the mixture three times in the microwave for one minute and stirred. We added a drop of ethidium bromure to the cassette in the dark (to preserve its dyeing ability), and when the agarose gel we prepared cooled down, we added it to the cassette. After waiting for half an hour for the gel to polymerize, we put the cassette in the tank, added the sterilized water, and then we added:

  1. Pet26
  2. Laccase
  3. Uncut pet26
  4. Uncut laccase.

We waited for 25 minutes for the DNA to walk.

Then we compared them in UV light.

Ligation

We mixed 4 microliters of buffer, 1 microliter of enzyme, 7 microliter of pet26, 3 microliter of laccase and 5 microliter of nuclease free water. And we stood at 16 degrees. Then we transformed them and checked them.

High Efficiency Transformation

We transferred gene to the bacteria because we want to reproducing the bacteria

Protocol

  1. Thaw a tube of SHuffle Competent E. coli cells on ice for 10 minutes
  2. Add 1–5 μl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipette 950 μl of room temperature SOC into the mixture.
  7. Place at 30°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 30°C.
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform several fold serial dilutions in SOC.
  10. Spread 50–100 μl of each dilution onto a selection plate and incubate overnight at 30°C. Alternatively, incubate at 25°C for 48 hours.

Expression Using SHuffle

We made gene expression for turning gene to the protein and for producing enzyme.

Protocol

  1. Transform expression plasmid into SHuffle. Plate on antibiotic selection plates and incubate 24 hours at 30°C
  2. Resuspend a single colony in 10 ml liquid medium with antibiotic.
  3. Incubate at 30°C until OD600 reaches 0.4–0.8.
  4. Add the appropriate inducer, e.g. 40 μl of a 100 mM stock of IPTG. Incubate for 4 hours at 30°C or 16°C
  5. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction alone.
  6. For large scale, innoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 30°C until reaches 0.4–0.8. Add the appropriate inducer, e.g. IPTG to 0.4 mM. Induce 4 hours or 16°C overnight.

Protein Isolation

E.coli SHuffle cells including the recombinant laccase gene were induced by IPTG overnight for expression of laccase enzymes.

After expression, polyhistidine tagged laccase enzymes were isolated with magnetic tube rack (GeneOn) and the MagneHis Protein Purification System (Promega).

Restriction Enzyme Digestion

We added restriction sites on the two ends of the laccase gene, which are necessary for the enzymes to cut the DNA and suitable for the expression vector.

While selecting the enzymes we were going to use, we paid attention that recognition sites of the enzymes are not within the laccase gene. In this way, we prevented the enzymes from cutting the gene and causing the gene's inability to function.

We used two different restriction enzymes to prevent the cut ends from being complementary and sticking to themselves.

We isolated the expression vector and laccase gene from cut products analyzed by agarose gel electrophoresis. After the density of the DNA extracted from the gel was determined, we ligated the vector and the gene to be cloned in a 3:1 ratio.

The Protocol

Day 4 : 20.07.2019

Plasmid Isolation Protocol with Kit

  1. Collection of bacteria: E.coli in 1.5 ml micro-centrifuge pellet is taken to overnight culture in 1.5-5 ml LB medium containing appropriate antibiotic.
    2. Note: Solution 1,2,3 allows cell membrane disintegration because it contains SDS, it breaks down the lipids in the cell membrane. It removes the proteins.
  2. Resuspension: We added 100 micro-liters of “solution 1 ”and we resuspended the bacterial palette with vortex.
  3. Lysis: We added 200 micro-liters of “solution 2”, and mixed slowly by inverting the micro-tubes 4-5 times, keeping it at room temperature for 1 minute. If “VisualLyse” is added, the solution turns blue, stirring slowly must be done until a homogeneous blue solution is obtained.
  4. Neutralization: We added 350 micro-liters of ‘’solution 3’’. We slowly mixed the micro-tube 4-6 times and let it stand at room temperature for 1 minute. If ‘’VisualLyse ”is added, mixing slowly must be done until all blue marks are removed.
  5. Pellet Cell residues are centrifuged for 5 minutes at 10000 rpm in micro-centrifuge.
  6. DNA binding is transferred to the supernatant spin column. It is then centrifuged for 2 minutes and the flowing liquid discarded. Special bit tube with supernatant spin column filter.
  7. Washing: 750 micro-liters of Solution Wash Solution is added and centrifuged for 1 minute and then the flowing liquid is discarded.
  8. Wash the Column again.
  9. Centrifuge for 1 minute to remove any “Wash Solution” residue.
  10. Elution: Transfer the column to a new 1.5 ml micro-tube. Add 50 micro-liters of Elution Buffer to the center of the Spin Column and centrifuge at 10,000 rpm for 2 minutes. The elution buffer washes the plasmid DNA in the filter.

Day 5 : 25.07.2019

Recovery from gel

(We will look at the gel to make sure there is a cut)

Weigh the gel (0.24 g) (we put 1000 times the amount of gel in it)

We thawed the gel in 240 microliters of DB buffer.

Spined the gel. The gel settled.

We melted the gel at 50 ° C.

We transferred the melted gel solution to the column and centrifuged at 10000 rpm for 1 minute and discarded the residue. (To get rid of DNA waste)

We washed the column with 650 milliliters of wash buffer.

We centrifuged it at 10000 rpm for 1 minute.

As a guarantee, we washed the column again with wash buffer and centrifuged again at 10000 rpm for 1 minute.

We centrifuged it at 10000rpm for 1 minute without adding buffer.

To precipitate the DNA we binded it to the column, we first added 30 microliters of nuclease free water to the column and then centrifuged it, then added 20 microliters and centrifuged it again (50 microliters in total). And we raised it to -20 degrees.

After cloning

We've isolated it to increase our DNA stock.

After expression

We ligated the DNA which came to us. Because that we ligate in small amounts, we cannot put them directly on the gel and control them. We cloned and amplified DNA into good bacteria, and isolated it. We first isolated it to check whether the laccase and the expression vector were correctly bounded and to check whether the laccase gene was cloned into this expression vector. We loaded the plasmid DNA on our gel without ever cutting it. We applied the cutting enzymes we used here to isolate the material and wait for it to cut. To control this, we isolated the DNA after expression and looked at the DNA in gel. We stocked some of our isolates and used them for control.

Purification with gel

In order to remove the useless part of the plasmid that came to us and to remove the unwanted parts in the vector, we first electrophoresed and separated these parts and also checked whether our segments were correct. If our cut is correct, we took the small part corresponding to the laccase and the big part corresponding to the expression vector, isolating them from the gel and connecting them together. Thus, the laccase was in the ve expression bacteria we wanted.

Procedure

After Expression

Before using, heat the lisis and neutralisation buffers at 37°C then wait for them to cool down at 25°C. Heat the elution buffer to 70 ° C and apply to column.

Collecting bacterias

Collect bacterial culture at 8000rpm (6800 x g) for 2 minutes in a centrifuge.

Carefully empty the supernatant and remove any residue.

Suspended cells, thawing, neutralization

To pellet cellules:

  • Add 250µL Resuspension Solution and vortex
  • Add 250µL Lysis Solution and turn the tube inside 4-6 times
  • Add 350µL Neutralization Solution and turn the tube inside 4-6 times

Centrifuge for 5 minutes

Linking DNA

Transfer supernatant to Thermo Scientific GeneJET Spin Column

Washing the column

Add 500µL Wash Solution and centrifuge for 30-60 seconds, then empty the liquid. ( x 2 )

Centrifuge the empty column for 1 minute.

DNA recovery from agarose ge

Gel electrophoresis

Run DNA Sample on agarose gel electrophoresis to seperate DNA fragments. Perform ethidium bromide staining for DNA visualization.

Cut agarose gel band containing the desired DNA and place it into a pre-weighed microcentrifuge tube.

Solubilization of agarose

Determine the nett weight of gel slice and add 1 volume of Buffer DB to 1 volume of gel.

Centrifuge the tube briefly to make sure the gel slice stays at the bottom of the tube.

Incubate at 50°C until gel has melted completely.

Mix occasionally to ensure complete solubilisation.

DNA purification

Loading to column

Transfer the sample into a column assembled in a clean collection tube. Centrifuge at 10,000 x g for 1 min. Discard flow through.

Repeat for any remaining sample from step 2.

Column washing

DWash the column with 650µl wash buffer and centrifuge at 10,000 x g for 1 min. Discard flow through.

Column drying

Centrifuge the column at 10,000 x g for 1 min to remove residual ethanol.

iGEM Team Saint Joseph

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