Team:RPI Troy NY/Description

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Project Description

♦Abstract♦

Sophorolipids are a group of amphipathic compounds synthesized by yeasts, including our selected chassis Starmerella bombicola. These compounds are among the most industrially viable biosurfactants. As surfactants, sophorolipids are superior to current synthetic alternatives largely due to their low toxicity and ready biodegradability. Anti-cell proliferation and anti-microbial assays suggest that medium-chain length sophorolipids possess therapeutic potential. However, their higher cost of production poses a major barrier to commercial use. Even when grown on a substrate largely composed of fatty acids, a significant portion of the fatty acids are diverted from sophorolipid production due to competition with the β-oxidation pathway. Fatty acids diverted into the β-oxidation pathway undergo irregular cleavage, lowering sophorolipid yield and creating a heterogeneous mixture of products. Full suppression of this pathway would result in undesirable metabolic disruption. Selective suppression of this pathway through genetic engineering could reduce production costs through improving the yield of useful chain lengths.

♦Design♦

In this project, we sought to increase the production of sophorolipids by taking a two-part approach. In the first part, we engineered primers and inserted these into the yeast to be able to produce either normal amounts of sophorolipids or lesser amounts of sophorolipids (gene knockout). In the second part, we extracted these sophorolipids and completed analysis on this.

Protocols

♦Microbial Genetics Protocols♦

Our Microbial Genetics group used PCR amplification and fusion PCR to construct our sequences with the modified genes. PCR amplification was used with primers designed to facilitate fusion PCR after the regions had been amplified. Primers were designed using Snapgene software and ordered from IDT Technologies. Melting temperatures of primers allowed for appropriate annealing and extension of the sequences during both the PCR amplification and fusion PCR phases. Stepwise fusion PCR allowed us to assemble the DNA parts in the appropriate order. The modified sequences were followed by a selection marker (hygromycin B gene) among additional terminators to allow for growth in the presence of antibiotic.

Agarose Gels

Analytical Gel

• 1-2% Agarose

• Ethidium Bromide (1uL/100mL)

Purification Gel

• .8% Agarose

• Ethidium Bromide (1uL/100mL)

PCR Amplification

• 50ng Template DNA

• 25uL Accuzyme or Hotstart Mix (1uL/100mL)

• 1uL primer stock (10mmol forward and reverse primer)

• Remainder MilliQ

• 1uL pure DMSO (Optional, use if high GC content)

50uL total

Fusion PCR

• 100ng each PCR amplicon (equimolar value)

• 25uL Accuzyme or Hotstart Mix (1uL/100mL)

• Remainder MilliQ

50uL total

Run 10 cycles of appropriate annealing/joining protocol in Thermalcycler

immediately add 1uL primer stock (10mmol forward and reverse primer)

Run 20 cycles of appropriate annealing amplification and extension protocol in Thermalcycler

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Gel Purification

BioRad Gel Extraction Kit

Gels purified on .8% agarose gels

♦Chemical and Culturing Protocols♦

We grew the yeast in two types of broth: FM2 and PM2. This is the components of each of those broths.
PM2 Ingredients Amt.(g/L) FM2 Ingredients Amt.(g/L)
Glucose 200 Glucose 100
Yeast Extract 5 Yeast Extract 5
KH2PO4 1 KH2PO4 1
MgSO4 0.5 MgSO4 0.5
CaCl2 0.1 CaCl2 0.1
NaCl 0.1 NaCl 0.1
Peptone 0.7 Peptone 0.7

Following steps might vary depending on number of samples.

Dissolve ingredients in Distilled Water

Glucose (dextrose) should NOT be added to the broths before autoclaving; glucose should be made separately (500 g/L), with a final pH of 3.5 will be added in proportion to the nutrient broths (see above for final concentration of ingredients) to the culturing/fermentation flasks.

Adjust pH of both PM2 and FM2 broth to 4.5

Autoclave at 121*C for 15 minutes (setting 4)

We grew the yeast on agar plates. This is the components of the agar.
PM2 Ingredients Amt.(g/L)
Agar 20
Yeast Extract 10
Glucose 100
Urea 1

Dissolve ingredients (except urea) in Distilled Water and mix with magnetic stir bar (leave stir bar in solution to help liquefy (with hot plate) agar excess after plating if it becomes solid).

Alternatively, microwave it for ~4 minutes, occasionally stopping microwave every ~30 seconds and swirling bottle to distribute heat evenly.

Autoclave at 121*C for 15 minutes (setting 4)

Sterilize Urea with urea powder on top of bottletop-filter (.2 micrometer pore length) and sterile DI water. Add sterile urea to agar under an active bunsen burner.

Pour 20-25 mL Agar on sterile plate under sterile conditions.

Inoculate plates with S. bombicola. Leave in incubator to grow for 48 hours at 30*C

With an active Bunsen burner, prepare 250 mL Erlenmeyer flask with 20 mL sterile glucose (500 g/mL), and 80 mL PM2

Add 1 colony of yeast (either WT or KO)

Incubate in a shaker incubator for 48 hours at 225 rpm at 30*C

Remove FM2 inoculum

With an active Bunsen burner, prepare 3 x sterile 100 mL Erlenmeyer flask with 44 mL FM2, 5mL sterile glucose (500 g/mL), 0.5 mL sterile oleic acid, and 0.5 mL of culture grown on an agar plate for each mutant/wild type.

Replace flasks in shaker incubator for 48 hours at 225 rpm at 30*C.

After 48 hours, under an active Bunsen burner., add 1 mL sterile Oleic acid.

Return to shaker incubator, 225 rpm, 30*C.

Do this for four consecutive days (96 total hours).

On the fifth day, remove from shaker, add 20 mL n-hexane, and put in freezer to kill yeast so sophorolipids will not be destroyed.

Take dry cell weight of the yeast by weighing sterile bottletop filter and running liquid through, then weighing filter again.

This is the protocol we used to extract the sophorolipids:

Rotovap

Add equal amounts of n-Hexane (50mL), wash twice to remove excess oleic acid. Sophorolipids will be on bottom layer.

Add equal amounts of Ethyl Acetate (50mL), wash twice to remove water.

Sophorolipids will be on the top layer of the addition flask, with the ethyl acetate (sophorolipids will be miscible in the ethyl acetate).

Place in 250 mL round bottom flask.

Rotovap around 50-60*C (warm water bath) ~90-120 rpm, around ~100 torr until liquid is ~20 mL.

Make sure to remove sophorolipids from the round bottom flask and transfer to a vial before sophorolipids adhere to the sides of the round bottom flask and make it difficult to remove for storage/weighing.

Weigh and label an empty glass vial, and put remainder liquid in it; use vial adapter to attach the vial to the rotovap and rotovap the remaining liquid.

Sophorolipids should have turned to a pale yellow to golden-brown color.

Weigh flask again to get sophorolipid weight.

Store in fridge.

Analyze using LCMS/NMR to get #of carbons in chain.

Notebook

♦Microbial Genetics Notebook♦

01OCT2017

First round of primers ordered

04OCT2017

Second round of primers ordered to accomodate correction in PreDver2_F

05OCT2017

Primers resuspended to 100mmol

Primer working stock made at 10mmol

First round of primers ordered

First round of primers ordered

See Cloning Protocol. No DMSO used. MgCl2 used with genomic DNA, TA=50C, Extension time=4 min

06OCT2017

Correct primer came in for PreDver2_F

Primers resuspended to 100mmol

Primer working stock made at 10mmol

PCR amplification of the following samples (in duplicate):

See Cloning Protocol. No DMSO used. MgCl2 used with genomic DNA, TA=50C, Extension time=2 min

07OCT2017

Relabeled samples for ease of use

No bands existed on the gel for samples A1, A2, G1,G2. PCR amplification rerun for each of these samples:

See Cloning Protocol. No DMSO used. MgCl2 used with genomic DNA, TA=50C, Extension time=2 min

Gel Purification- .08% Gel (eluted from columns with 30uL milliQ)

08OCT2017

2% analytical gel for PCR product of A1,A2,G1,G2

Smudge for A1&A2; nothing present for G1&G2 (same result when run on .08% gel)

Followed up with Jian- as it turns out, we can’t run an analytical gel on a piece as small as A (PreDA ~30bp)

However, had to rerun PCR on G1&G2. This time, using TA=47C and Extension time of 1:30

We will simply use the A1 or A2 PCR product for the fusion PCR reactions. The PCR product has been transferred to 1.5mL

2% analytical gel for PCR product of G1, G

No bands observed

09OCT2017

Errors discovered in primers, Jian has designed 3 new ones and will send the sequences to Krystyna to order

note: Fusion PCR is still likely possible with PreA/PostA and PreB/PostB

Fusion PCR conducted on above constructs

11OCT2017

Ran gels and gel purified PreA/PostA and PreB/PostB fusion products

Prepped two A samples and sent for sequencing

15OCT2017

Sequencing came back positive for domain A KO region

New primers came in from IDT

Resuspended to 100mmol

Made 10mmol stock

PCR amplified Term1 and End with new primers

Term1 worked, extremely faint bands found on End lanes, but in the correct position

Repeated End PCR with 1uL PCR product as template

Worked very welll

Gel purified Term1 and End

Fusion PCR of Term1/HygB

16OCT2017

Analytical gel of Term1/HygB

Part appears small? Maybe still starting material?

STOP, REPEAT FUSION PCR

Still unsuccessful

17OCT2017

Blossom reran PCR on HygB with Jian

Ran visual and purification gels on HygB

22OCT2017

Term1/HygB gel purified from Jian’s gel samples

Concentration is 12.44 ng/microL

Ran fusion PCR with Term1/HygB + End

♦Chemical and Culturing Notebook♦

The Culturing committee chose to use handwritten documentation in a notebook to record activities done by lab members on a day-to-day basis. Here are some examples of these pages.

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