Team:NAWI Graz/Protocols

Preliminary tests

To determine the viability of our project idea we started by conducting several tests:

We chose 2 strands of yeast as potential tools for esterification of our selected fatty acids (FAs):

BY4742 WT (wild type)
BY4742 elo10 (knockout mutant)

Linoleic acid (FA C12:0)
Myristic acid (FA C14:0)

12,1mg C14
28,2mg C12

We created a simple YPD medium for main-cultures with the following recipe

And diluted 12mg of linoleic acid and 28mg of myristic acid in 1mL of EtOH (FA-mix).

We started cell cultivation under 6 different conditions:

100 mL of YPD medium was filled into an Erlenmeyer shake flask for each sample. Samples 1), 2), 4) and 5) all where supplemented with 250µL of FA-mix (10mg FAs per 100mL C12:C14 in a 3:1 ratio)
For samples 1) and 4) 1g of the detergent Brij58 was added to improve dilution of the hydrophobic FAs.
Samples 3) and 6) will receive the same amount and ratio of the FAs after the glucose in the medium should be completely used up. This should be at a cell count of about 1.5*10⁸.

The inoculated amount from the ONC (over night culture) was chosen to roughly fit this cell count and our time table. All samples where supplemented the same amount of ONC (15µL).

Those prepared sample media where then autoclaved for following inoculation with the yeast strains.

Mass balance:

Samples 3) and 6) will receive the same amount and ratio of the FAs after the glucose in the medium is predicted to be completely used up. This should be at a cell count of about 1.5*10⁸. The inoculated amount from the ONC was chosen to roughly fit this cell count and our time table. All samples where supplemented the same amount of ONC (15µL). The samples where then put in to the shaker for 30°C and then shaken at about 200 rpm.

As planned we supplemented samples 3) and 6) with 100µL of FA-mix. (14mg C12 + 6mg C14 in 200µL EtOH).

We also observed that samples 2) and 5) (the samples that already had been supplemented with FAs but no detergent was added) showed no visible cell growth. We suspected that the stress from the FAs in the medium put too much stress on the yeast cells and subsequently they died off. We tried to find some alive cells under the microscope, but none could be seen. Nonetheless we kept both samples [ 2) and 5) ] in the shaker in the hopes of some sort of progress the next day.

To observe which one of the FAs could have prevented cell growth we added two additional samples,
7)WT+C12 and 8)WT+C14, with our already prepared medium to the shaker.

A few hours later the progress of samples 7) and 8) was checked, with sample 8) already showing new cell growth. Sample 7) was visually undistinguishable from before.
Cultures 1), 3), 4) and 6) were harvested by centrifugation and throwing away the supernatant. The cell pellets were shock frozen with liquid nitrogen and put into a -80° cooler.

The cell count of each sample was determined by a CASEY:
Dilution: sample:caseyton 1:5000

Samples 2) and 5), whose yeast cells seemed to have died the day before, accumulated living cells overnight. The samples 2), 5), 7) and 8) were given additional time to grow cells and kept in the shaker.

The remaining samples where harvested the same way as the previous ones and counted with the CASEY.

Samples 2), 5), 7) and 8) were also streaked on Agar-Agar plates with YPD medium to check if the growth delay for following cultures persists (mutation or persistent delay due to FA supplements). A light microscope was used to check if the cells were still healthy.

The streaked-out cultures were used to prepare ONCs so we could check with another cultivation batch for mutations in our yeast strains. (test lipid extraction)

The following protocol was used for lipid extraction of the samples 1) WT+D+FA, 2) WT+FA, 4) ELO+D+FA, 5) ELO+FA and 6) ELO

For isolating the triacylglycerols, we used TLC. Since we used a sampler to apply our samples we only applied 60% of our total lipid extract. As solvent for the TLC we used Petrolether:Diethylether:acetic acid 32:8:0,4. Staining was done with an iodine chamber and the triacylglycerol spots were marked.

The triacylglycerol spots where scratched off the TLC and treated with the following protocol

These results led us to divert our focus away from yeast, instead we focused in achieving better results in E. coli.

Project implementation

As first step we wanted to combine the gBlocks to the full expression cassette in a series of OE-PCR reactions like seen in Figure 8. For the OE-PCRs the “high fidelity phusion polymerase kit” from thermofisher was used with the IDT synthesized gBlocks that were to be combined. For amplification of the OE-PCR products, fitting primers were also ordered from IDT.. Both - gBlocks and primers - were prepared as suggested by thermo fisher in order to use them for the following OE-PCRs.

The table shows the exact nature of the reaction mixes used for the first OE-PCR reactions (Tab. 4.1.7). The respective primers and templates were chosen to create gBlock1+2, gBlock3+4, gBlock5+6, gBlock7+8 and gBlock 9+10.

For the thermocycler the program below (Tab. 4.1.8) was used. Annealing temp was chosen at 58°C since it is the melting temperature of the primer with the lowest melting temperature.

To check the results of this reaction an Agarose gel-electrophoresis was used:

Since EtBr is used for the gels, all work with gels is to be strictly kept in the designated lab. This avoids contaminating other workspaces with EtBr and increase workplace safety.

For the gel electrophoresis 2 stock solutions were created:
20ml TeA in 1L ddH2O - gel stock solution
20ml TeA in 2L ddH2O - buffer stock solution

Gels were cast by weighing 0.5g of Agarose and dissolving it in an erlenmeyer flask with 50ml of the gel stock by use of a microwave (with close attention to boiling retardation). The solution was then cooled under running water and poured into the cast. One drop of EtBr was added and the solution was mixed gently with the comb. The comb was then placed in the upper third of the gel until it hardened (about 20min). After the gel set, it was transferred from the cast into the electrophoresis chamber and filled the chamber with the buffer stock solution until the gel was covered with it. The PCR samples were then supplemented with 2µL of 4x loading dye and filled into the slots created by the comb, the last slot was filled with a standard solution for comparison with our results. The electrophoresis was then started with a voltage of 80V and kept running for about 1h 30min or until the dye front was well over the middle of the gel. Gels were prepared and handled the same way for all following agarose gels needed. We planned to cut out the successful bands but realized that no useable bands where present and made a photo of the gel for documentation (Fig. 4.1.7).

Since no gBlock showed a band at the respective height for the combined sequence, a rework of our OE-PCR protocol was in order.

To improve our method we prepared different mixes of the gBlock1+2 OE-PCR but under varying conditions. (Tab. 4.1.9)

The settings for the thermocycler were kept the same as the last time, since the strong suspicion arose that the missing DMSO caused the first reaction to fail (Tab. 4.1.8).

The gel showed that the PCR-reactions without DMSO did not work for our purposes making it mandatory to be supplemented. The mix with a bigger amount of primer showed worse results than the mix with the original concentration. Comparison with the bands of the standard revealed that the bright bands of our samples did not belong to the wanted combined gBlock1+2 but very possibly to the uncombined gBlocks (Fig. 4.1.8).

To improve the results of our OE-PCR we again consulted the professionals at ACIB, who helped us in making a new OE-PCR protocol to fit to our conditions.

For the new reaction we split the OE-PCR in two reactions, one just for the elongation of our overlapping templates (Tab. 4.2.1 upper part) and one for the amplification of the combined sequence (Tab. 4.2.1 down part). After the first reaction was done the second reaction mix was added to it for a full volume of 50µL. The thermocycler program was also changed to a reduced annealing temp. of 54°C. We applied this method to PCR mixes for gBlock1+2, gBlock3+4, gBlock5+6, gBlock7+8 and gBlock9+10.

The resulting gel showed us that our results improved vastly, but only a few of the gBlock combinations yielded a satisfying amount of product (Fig. 4.1.9).

For the sequences with enough product, the next step OE-PCR was done yielding only very limited success. It was suspected that another type of polymerase could be useful for those reactions but a lack of resources led to a reconsideration of the used approach to the project.

As a result, the following step was the OE-PCR of the 2 parts from the mutated tesA cassette instead of the much longer esterification cassette. The used method was exactly the same one as used for the last gel (Tab. 4.2.1) with the result of bright and strong bands at the expected heights in respect to the standard (Fig. 4.2.1). A gel purification kit from thermofisher was used to extract the wanted sequence from the gel for the subsequent integration into the pTargetF vector via restriction digest and following ligase.

Stearic acid synthesis in a yeast delta-desaturase strain

Through interviews with industry professionals the project gained an additional goal. Since not the final esterified triacylglycerol is the important product for many companies, the new task was the biosynthesis of stearic acid as needed for the wax industry. This new assignment was approached by growing a yeast delta desaturase strain by supplementing it with oleic acid (10 mg/L and 40 mg/L) and following lipid extraction. For this matter, the same steps as in the preliminary tests were taken, so a comparison to the data we acquired there can be drawn.

The strain was visibly more red than the strains from the preliminary test, the colour also intensified with less oleic acid added (Fig. 4.2.2).

A light microscope was used to see if the cells were still healthy because of the strange color:

The TLC for after the lipid extraction showed that the triacylglycerol band for the tested strain ran further up the plate than the applied standards bands. It was suspected that this occurred because of the differing fatty acids the triacylglycerols are composed of. The slight drag downwards of the bands is a sign of plate oversaturation (Fig. 4.2.2).

The delta desaturase esterified a lot of the oleic acid for both supplemented concentrations. The strand with less supplemented oleic acid (10 mg/L) also esterified less of it and more of the wanted stearic acid. With the larger supplemented amount (40 mg/L) of oleic acid, the quantity of esterified stearic acid was even lower than the wild type or the elongase knockout strain.