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The objective of the lab course is to construct a new plasmid cloning vector called pTA7. This vector will be an E. coli / Saccharomyces cerevisiae shuttle vector. This vector will be useful for cloning using the Yeast Pathway Kit, see the next section "Background" "for more details.
In the mec research group, we are interested in understanding and engineering the biosynthesis of fatty acids and related products by the unicellular fungi known as baker's yeast S. cerevisiae.
Genetic engineering of complex traits require the simultaneous deletion and/or expression of multiple genes. This is a challenging problem as genetic engineering is time consuming. To solve this problem, we developed a protocol for the assembly of metabolic pathways that we call the Yeast Pathway Kit (YPK). See our publication in ACS Synthetic Biology for more details.
We use this protocol for quick construction and expression of large metabolic pathways in baker's yeast Saccharomyces cerevisiae such as this heterologous fatty acid synthesis pathway. Plasmids such as the one we will construct in this lab course are used to propagate these constructs in E. coli or S. cerevisiae.
In order to do this they need at least:
- a selection marker for E. coli
- a selection marker for S. cerevisiae.
- an origin of replication for E. coli
- an origin of replication for S. cerevisiae.
The first plasmid we used for this purpose in YPK was called pYPKpw and it has the following functional parts (Table#1):
| Table#1, pYPKpw | part | function |
|---|---|---|
| ampR | selection marker for E. coli. | |
| pUC | origin of replication for E. coli. | |
| 2µ | multicopy origin of replication for S. cerevisiae from the natural 2µ plasmid | |
| URA3 | selection marker for for S. cerevisiae. | |
| Δcrp | a partial, inactive E. coli cyclic AMP receptor protein or CRP gene. |
The Δcrp is an E. coli gene which is inactive and only provide a recombination site. The pathways that we make are meant for S. cerevisiae, but we often need to transfer the pathway to E. coli so we can obtain larger amounts of higher quality DNA for analysis or transformation.
The pUC origin of replication (ORI) results in a high copy number of the vector in E. coli which is an advantage for obtaining large amounts of DNA. However, we have observed genetic instability in E. coli for some large pathways that we suspect is linked to high copy number. Our experience is that a lower copy number provides more stability.
We conceived a series of plasmid vectors called pTAx where x is a number from 1 to 11 (at the moment). The pTAx vectors were designed to have a lower copy number in E. coli to try to solve the stability problems of pYPKpw. The pTAx plasmids should have a lower copy number in E. coli than the pUC based pYPKpw since they have the pBR origin of replication that includes the ROP gene. The first pTAx plasmid, pTA1 was constructed by a former post-doc in the group, Tatiana Andrevna, hence the name.
The pTAx vectors are made from five genetic elements (Table #2), see (pTAx assembly strategy).
Each element is a distinct segment from a particular source plasmid. The pTAx vectors are similar to each other, but differ in the selection markers (yeast marker) and yeast origin of replication (yeast ORI).
| Table#2 | Name | E. coli marker | E. coli ORI | yeast ORI | yeast marker | MCS | Constructed by: | Enzyme to linearize | 🥶 freezer list number(s) | Sequenced? | Date |
|---|---|---|---|---|---|---|---|---|---|---|---|
| pTA1 | ampR | pBR | 2µ | LEU2 | Δcrp | Tatiana Pozdniakova | AatII, ZraI, FspAI | µ828, µ928, µ929 | ✅ | 2019-10-xx | |
| pTA2 | -"- | -"- | CEN/ARS | LEU2 | -"- | EGB2023 | AatII, ZraI, FspAI | µ1814, µ1815, µ1817 | 2023-06-01 | ||
| pTA3 | -"- | -"- | 2µ | HIS3 | -"- | Tatiana Pozdniakova | AatII, ZraI, FspAI, EcoRV | µ1271 | 2021-07-09 | ||
| pTA4 | -"- | -"- | CEN/ARS | HIS3 | -"- | EGB2023 | AatII, ZraI, FspAI, EcoRV | µ1816, µ1818, µ1819 | 2023-06-01 | ||
| pTA5 | -"- | -"- | 2µ | KanMX4 | -"- | Paulo Silva, Julio Freire | AatII, ZraI, FspAI, EcoRV | µ1652 | ✅ | ||
| pTA6 | -"- | -"- | CEN/ARS | KanMX4 | -"- | GMB20 | AatII, ZraI, FspAI, EcoRV | µ520 | 2020-12-23 | ||
| 🔥 | pTA7 | -"- | -"- | 2µ | TRP1 | -"- | EGB2025 ❓ | AatII, ZraI, FspAI | ❓ | ❓ | ❓ |
| pTA8 | -"- | -"- | CEN/ARS | TRP1 | -"- | GMB20 | AatII, ZraI, FspAI | µ521, µ522 | 2020-12-23 | ||
| pTA9 | -"- | -"- | 2µ | URA3 | -"- | Tatiana Pozdniakova | AatII, ZraI, FspAI | µ1272 | 2021-07-09 | ||
| pTA10 | -"- | -"- | CEN/ARS | URA3 | -"- | GMB20 | AatII, ZraI, FspAI | µ523 | 2020-12-23 | ||
| pTA11 | -"- | -"- | 2µ | LEU2d | -"- | EGB2024 | AatII, ZraI, FspAI | µ542 | ✅ | 2024-05-23 |
The lab course is divided into nine practical classes. Each student attends three of the nine classes.
| LAB | Task | ||||
|---|---|---|---|---|---|
| 1️⃣ | PL1 | Prepare plasmid DNA from E. coli (miniprep) | LAB1 | ||
| 2️⃣ | PL2 | Analyze plasmid DNA by agarose gel ➕ Prepare PCR reactions for each plasmid element | [[LAB2 | ||
| 3️⃣ | PL3 | Analyze PCR products by agarose gel ➕ Inoculate S. cerevisiae for transformation | [[LAB3 | ||
| 4️⃣ | PL1 | S. cerevisiae transformation | [[LAB4 | ||
| 5️⃣ | PL2 | In-silico assembly of plasmid 💻 | [[LAB5 | ||
| 6️⃣ | PL3 | S. cerevisiae colony PCR | [[LAB6 | ||
| 7️⃣ | PL1 | Analyze Colony PCR products by agarose gel ➕ Prepare solid LB medium for LAB8 | [[LAB7 | ||
| 8️⃣ | PL2 | Yeast DNA preparation ➕ E. coli transformation (Plasmid rescue) | [[LAB9 | ||
| 9️⃣ | PL3 | Plasmid alkaline lysis miniprep with commercial kit. | [[LAB9 | ||
| 🔟 | (Opt) | (Opt) | (Opt) | Analytical restriction digestion | [[LAB10 |