Re-integrating a dry scoby into a new culture

 

November 10

November 15

Venus

 

Synscoby Co Culture

Day 0 (Wednesday PM) November 30th

1. Inoculate K. rhaeticus from glycerol stock in 5mL YPD + 1% cellulase
2. Place in shaking incubator at 30˚, 250rpm for 3 days
3. Streak S. cerevisae on YPD plates so fresh when inoculating on day 3.

To avoid contamination the YPD + Cellulase 1% were added into a centrifuge tube in the hood, then added to smaller to tubes with snap off caps.

Cellulase                                                    YPD

Outside of the hood we added the K.Rhaeticus with tooth picks into 30 ml tubes.

There were a total of 6 tubes so we can start co coulture of (3x BY4741p + 3 x Venus Ura)

Streaking new fresh yeast from the October batch into Agar plates 

Isolating yeast at this point is as simple as taking a very small amount of your culture and rubbing (streaking) it on to an agar plate. Because of the stable, non-liquid agar medium, once streaked, single colonies of microbes are essentially stranded by themselves.

After a few days or weeks (depending on incubation temperature and microbe population), they’ll multiply and grow large enough to be seen with the naked eye. At that point, it’s a matter of selecting the colonies you like and growing them up to larger amounts.

It’s impossible to stress enough how important cleanliness and sanitation are at this point in the process. If you truly want a pure strain, you need to ensure you’re not contaminating what you’re trying to isolate with other cultures.

Day 2 (Friday PM) December 2nd

1. Inoculate S. cerevisiae from plate in 5mL YPD
2. Place in shaking incubator at 30˚, 250rpm for 1 day

Day 3 (Saturday) December 3rd

1. Centrifuge K. rhaeticus at 3,220 g  (5119 rpm) for 10 minutes. 
2. Resuspend K. rhaeticus cells in YPS to OD600=2.5. This helps to remove cellulase.
3. Dilute S. cerevisiae in YPS to OD600 = 0.01
4. Combine K. rhaeticus and S. cerevisiae in 50mm petri dish, inoculating K. rhaeticus 1/50 and S. cerevisiae 1/100 in fresh YPS-optiprep. Final volume should be 15mL.
5. Incubate at 30˚ for 3 days under static conditions. Do not move co-cultures while growing or BC layers will come apart.

*****

YPS

• 5g yeast extract

• 10g peptone

• 10g sucrose

HS Recipe x 500 ml

• 2.5g yeast extract

• 2.5g peptone

• 1.35g sodium phosphate

• 0.75g citric acid

• 10g sucrose

• 500ml distilled water

**Snap off cap works best for K. Rhaeticus growth 

K.Rhaeticus

Growing critters ins Hestrin Schramm HS {brown media)  (look recipe)

K. Rhaeticus (bacteria) :  HS (hestrin schramm)  30c

HS Recipe x 500 ml

HS 

• 2.5g yeast extract
• 2.5g peptone
• 1.35g sodium phosphate
• 0.75g citric acid
• 10g sucrose
• 500ml distilled water

---------------

Pour from the stock to a separate tube is done in the sterile hood 

Streaking from Plates. with the microbes from (Part1) : 

Taking a loopful of microbe culture (a colony) from the plates and putting them in Media

 

October 25 LEFT                                       November 2nd RIGHT pellicle is visible - grew in 30C shaker

   

November 2nd

We run a test to see which tube top worked better a snap off cap and a screw top

The Snap off seems to be better as it lets more air come in

November 4th - moving forward we will only use Snap off tops

Nov7 - Testing to see if the K. Rhaeticus we took from the frozen stock will grow on HS media

We just took a bit from the frozen stock that we put in the -80C

Growing the pellicle in motion or still. Both will be growing at 30C, One in the shaker and one in the incubator

Nov 14 , The one on the shaker grew, the one in the incubator did not grew.

Synscoby Part 3 Mini Prep : Plasmid purification

Only to purify Plasmid

Mini-Prep procedure is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis, sequencing, cloning, or other purposes.

The Monarch Plasmid Miniprep Kit is a rapid and reliable method for the purification of up to 20 μg of high quality plasmid DNA. (Protocol -tips).

The kit consist on the following steps:

1. PELLET  -  Spinning them fast to the cells sink and liquid media stays on top
2. RESUSPEND -  resuspend cells in first buffer
3. LYSE Cell lysis or cellular disruption is a method in which the outer boundary or cell membrane is broken down or destroyed in order to release inter-cellular materials such as DNA, RNA, protein or organelles from a cell. 
4. NEUTRALIZE - 
5. BIND -  Binds DNA to spin column (mini tube)
6. WASH -  to remove any last proteins, to keep the wanted DNA
7. ELUTE - Add a little salt to clean the DNA off the column and put it into a new tube

**QUBIT : Not from the Kit but a very important step. This will help determine the concentration, yield and purity of a DNA sample.  

• Needed for QUBIT: 
• MASTER MIX   ------> (Buffer + Regeant ) **Need to do calculations based on the amount of samples
• Qubit Assay tubes -  they do not absorb UV light . Must be used to get an accurate reading from the Qubit machine

How many samples do we have ?

Amount of Samples    N = 2

                                  Buffer = 200(N +2.5)

                                             200(4.5)

                                   Buffer =900  μL (microliters)

                                  

                                 Interagent = N+2.5

                                                    4.5  μL (microliters)

The term ‘plasmid’ was coined by Joshua Lederberg in 1952. Originally evolved from bacteria, plasmids are extrachromosomal genetic elements present in most species of Archae, Eukarya and Eubacteria that can replicate independently. Plasmids are circular double stranded DNA molecule that are distinct from the cells chromosomal DNA.

The structure and function of a bacterial cell is directed by the genetic material contained within the chromosomal DNA. In some cases plasmids are generally not essential for the survival of the host bacterium. Although not essential, plasmids contribute significantly to bacterial genetic diversity and plasticity by encoding functions that might not be specified by the bacterial chromosomal DNA. Plasmids specify traits that allow the host to persist in environments that would otherwise be either lethal or restrictive for growth. For example antibiotic resistance and protein expression. Antibiotic resistance genes are often encoded by the plasmid, which allows the bacteria to persist in an antibiotic containing environment, thereby providing the bacterium with a competitive advantage over antibiotic-sensitive species. As a tool, plasmids can be modified to express the protein of interest (e.g., production of human insulin using recombinant DNA technology).

Plasmids have served as invaluable model systems for the study of processes such as DNA replication, segregation, conjugation, and evolution. Plasmids have been pivotal to modern recombinant DNA technology as a tool in gene-cloning and as a vehicle for gene-expression.

Characteristics of Plasmid - Plasmids present in the bacterium differ in their physical properties such as in size (kbp), geometry and copy number.

Plasmid Size - Plasmids range in size from 1 kbp (kilo base pair) to 1000 (kilo base pair) megaplasmids that are many hundred base pairs in size.

Plasmid Geometry -Although most plasmids possess a circular geometry, there are now many examples of plasmids that are linear in a variety of bacteria. Plasmid DNA may appear in one of the five conformations nicked open circular DNA which has one strand cut, relaxed circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed, linear DNA has free ends, supercoiled DNA is fully intact with both strands uncut, and supercoiled denatured DNA is like super coiled DNA, but has unpaired regions that make it slightly less compact.

Plasmid Copy Numbers - Copy number refers to the average or expected number of copies per host cell. Plasmids are either low, medium or high copy number. Knowing which category plasmid falls under is very important when starting out an experiment. If working with a low-copy number plasmid which is associated with a low yield and might therefore be required to set up more cultures. On the other hand, if a poor yield is obtained from a high copy plasmid, troubleshooting is required. In bacterium with high copy number plasmids, during cell division the plasmids get segregate randomly in the daughter cells, whereas bacterium with low copy numbers, during cell division and partition the plasmids divided equally in the daughter cells. An advantage of high copy number is the greater stability of the plasmid when random partitioning (i.e. partitioning of plasmids into daughter cells) occurs at cell division.

Plasmid Isolation - The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations require the isolation of high purity plasmid DNA. The purified plasmid DNA can be used for immediate use in all molecular biology procedures such as digestion with restriction enzymes, cloning, PCR, transfection, in vitro translation, blotting and sequencing.

Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.

Sources:

https://www.mybiosource.com/learn/testing-procedures/plasmid-isolation

SynSCOBY Part 2 Growing Media E.coli

 October 25

Growing critters ins 2 media (LB, YPD)

• PNC (e.coli, plasmid  : LB. 37C
• PNS (e.coli, plasmid) : LB 37C
• BY4741(yeast) : YPD             30c
• VENUS (uria) (yeast): YPD.  30c
• 
  

Pour from the stock to a separate tube is done in the sterile hood 

Streaking from Plates. with the microbes from (Part1) : 

Taking a loopful of microbe culture (a colony) from the plates and putting them in Media

All Microbes grown overnight in Liquid Media

• PNC (e.coli, plasmid  : LB. 37C
• PNS (e.coli, plasmid) : LB 37C
• BY4741(yeast) : YPD             30c
• VENUS (uria) (yeast): YPD.  30c

For all microbes make glycerol stock 

60% glycerol to start and mix 50/50 with microbe culture. 30% Final

Store at -80C

JUST FOR E.COLI - 

Using LB + KAN

KAN is only for E.coli - use at 50-ug/mL

*Storing at -20oC and avoiding repeated freeze/thaw cycles will keep most antibiotics viable for at least 6 months.

Adding an antibiotic resistance gene to the plasmid solves both problems at once – it allows a us to easily detect plasmid-containing bacteria when the cells are grown on selective media, and provides those bacteria with a pressure to keep the plasmid

October 26

After being left overnight E.Coli s

YEAST ONLY

For all microbes make glycerol stock 

60% glycerol to start and mix 50/50 with microbe culture. 30% Final

Store at -80C

GROWS IN YPD in 30C

is placed on the 30oC 

Next Day (October 26)

Scoby with Matcha powder

 
After dying the bacterial cellulose with food dye and Butterfly Pea Flower Tea I was hoping to see if a high grade Matcha (green tea) could have a similar effect.

My past experiment make me believe caffeine is good for fermentation. I noticed adding some coffee powder actually helps. I was surprised the the cellulose turned wait not green.

I think it would be best to try again with a little "scoby mother" and see if the pellicle actual changes color.

I left it drying October 20, will report back as soon as it dries

Fully dry sheet.