Part:BBa_K5409711
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RPL3t(Terminators of yeast)
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RPL3t(Terminators of yeast)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Source
In order to obtain RPL3t in yeast, we first need to obtain the genome information of RPL3. The gene information of RPL3 can be retrieved at https://yeastgenome.org (SGD ID: S000005589). After downloading the target information(.fasta), identify the target ORF (open reading frame) and select the 400bp DNA sequence after the stop codon as our RPL3t terminator sequence.
Ribosomal 60S subunit protein L3; homologous to mammalian ribosomal protein L3 and bacterial L3; plays an important role in function of eIF5B in stimulating 3' end processing of 18S rRNA in context of 80S ribosomes that have not yet engaged in translation; involved in replication and maintenance of killer double stranded RNA virus.
Constructing
Based on the above 400 bp sequence, it is necessary to design primers (recommended length is about 20 bp) and amplify the target gene fragment by PCR in Saccharomyces cerevisiae. Agarose gel electrophoresis is then used to verify whether the target fragment size is correct.
Different promoters and terminators were assembled onto the ends of the ndps1 and ls target fragments. Since there are multiple PTPT combinations, here we take PTPT = (CCW12p, RPL3t, TDH3p, FBA1t) as an example. Primers were designed based on the PTPT combination (with correct homologous arms). Six fragments were amplified using PrimeStar Mix polymerase. CCW12p, ndps1, and RPL3t were assembled by homologous recombination, as were TDH3p, ls, and FBA1t, resulting in two fragments. The two fragments were then recombined to form one.
Next, we need to obtain the backbone of plasmid 352. Using YEp352 as a template, the Backbone-F/R primers and PrimeStar Mix polymerase were used to perform PCR, yielding a linearized YEp352 backbone.
The CCW12p-ndps1-RPL3t-TDH3p-ls-FBA1t fragment was recombined with the linearized YEp352 backbone through homologous recombination, and the resulting product was transformed into E. coli. Positive transformants from the Patch (A+) plate were selected and cultured overnight in LB medium(A+), after which the plasmid was extracted. The plasmid was then transformed into yeast, and successfully transformed yeast was activated in SDΔU. Activated yeast was preserved in 60% glycerol. (Refer to the protocol for transformation, LB (A+) medium preparation, plasmid extraction, PCR amplification, PCR product purification and homologous recombination).
Different promoter-terminator combinations in dual expression cassettes
Results of the agarose gel electrophoresis after PCR amplification of RPL3t from the yeast genome
Testing
After transforming them into yeast, shake flask fermentation tests were conducted. Since limonene is sparingly soluble in water (13.8 mg/L) and highly volatile, we adopted a two-phase fermentation system consisting of 10 mL SDΔU and 2 mL IPM (isopropyl myristate). IPM is a colorless, transparent oily liquid, insoluble in water, and capable of absorbing volatile limonene, which facilitates subsequent gas-phase detection. The culture conditions were 30°C, 220 rpm for 48 hours with an initial OD of 0.05. (Refer to the protocol for SDΔU medium preparation, gas-phase detection methods, and the creation of a standard limonene content curve).The strain we used for fermentation is (S. cerevisiae CEN.PK2-1C ΔROX1::tHMG1,IDI1)
The results of data are normalized
Measurement method
Gas Chromatography-Flame Ionization Detection(GC-FID)
Quantitative analysis of limonene in the product was conducted using a Shimadzu GC-2014C Gas Chromatograph, equipped with a flame ionization detector (FID). The chromatographic column used was an Agilent HP-5 capillary column (5% Ph-Me silicone, 30 m × 0.320 mm × 0.25 μm), with nitrogen as the carrier gas. (1) GC-FID Detection Method: The program settings were as follows: set the autosampler for automatic injection with an injection volume of 1 μL, using the split mode with a split ratio of 15:1. The detector temperature was set to 300°C, and the injection port temperature was set to 250°C. The column temperature was raised to 200°C at a rate of 30°C/min and held for 3 min, with a total run time of 20.91 min. (2) Preparation of the Limonene Standard Curve: Take 40 μL of 100 mM limonene stock solution into a 2 mL centrifuge tube, add 1960 μL of ethyl acetate to dilute it to 2 mM, and mix thoroughly. Prepare a series of standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM from the 2 mM limonene stock solution. Filter the solutions through a 0.22 μm filter membrane into chromatographic vials. After gas chromatography detection, record the peak areas of limonene and plot the standard curve. (3) Detection of Fermentation Samples: Take the recovered organic phase sample from the fermentation broth into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm for 1 min, and transfer 300 μL of the supernatant into a clean EP tube. Add 300 μL of ethyl acetate (EtAc), mix thoroughly, and filter through a 0.22 μm membrane into a GC vial. After GC detection, record the peak area and calculate the yield of limonene based on the standard curve.
Summary
After we constructed RPL3t into the CCW12p-ndps1-RPL3t-TDH3p-ls-FBA1t expression cassette, we tested it in the S. cerevisiae CEN.PK2-1C(ΔROX1::tHMG1,IDI1)and found that RPL3t worked properly.
Our work contributed new terminators to future teams (we can not find information about RPL3 terminators in iGEM Parts). However, we did not test the performance of RPL3t further and systematically. If there are iGEM teams interested in RPL3t, they can further explore its performance in different scenarios.
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