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Team:BostonU/Backbones
From 2014.igem.org
| The work carried out by previous BU iGEM teams used destination vectors, or plasmid backbones, with high-copy origins of replication. These high-copy plasmids in our library would not allow for optimal performance of our multiplexed transcriptional units and larger constructs. Additionally, works upon which we are basing our complex circuit assembly do not use high copy origins in their devices due to this loss of correct functionality. Circuit behavior would be desynchronized with the presence of a high copy origin, as it causes overexpression of the plasmid in a cell, leading to a high amount of transcription and protein expression. For more complex circuits, this overexpression pushes the limit of the amount of ribosomes that can be sequestered for translation, in addition to straining the cell's protein degradation mechanisms. This notebook details the process undertaken to replace the high copy pMB1 origin in our existing Level 1 and Level 2 destination vectors (named DVL1 and DVL2, respectively) with lower copy origins. Namely, the ColE1 (~50 plasmids/cell), p15A (~10 plasmids/cell), and pSC101 (~5 plasmids/cell) origins were selected to replace the high copy origin in DVL1 and DVL2. |
 Plasmid map of a MoClo Level 1 destination vector with original pMB1 origin of replication, LacZ fragment, and designed primers for backbone extraction. |
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June |
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Week of June 23 |
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| The backbones that would have their origin replaced were selected and new origins were selected. DVL1 with "A" and "E" MoClo fusion sites and DVL2 with "A" and "F" fusion sites were initially chosen, as they are the most commonly used MoClo level 1 and 2 destination vectors, respectively (See MoClo for more information on our assembly method). The general plan to replace the backbones was formulated, which comprised of: 1. Using PCR to extract the backbones without their high-copy origins from their full destination vectors, and to extract the lower-copy origins from their respective plasmids. 2.Performing a restriction digest on the backbone and origin fragments to have compatible sticky ends. 3. Ligating the origins to the destination vectors. 4. Transforming into E. Coli, purifying the plasmid DNA, and sequencing for confirmation. The PCR primer design added restriction sites for the MfeI restriction enzyme, which would give the ends of each of the amplified fragments compatible 4bp overhangs suitable for ligation. (Detailed primer design available here). All protocols used in this notebook are found in our protocols section. • Struck out devices with low copy origins for PCR on plates with appropriate antibiotic. • Prepared liquid cultures, incubated, and miniprepped. • Received primers, diluted. |
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Week of June 30 |
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• Carried out PCR of DVL1_AE, DVL2_AF, ColE1, p15A, and pSC101. • Ran gel to confirm primer functionality.  Lanes from left to right are: 2-log ladder, DVL1_AE, DVL2_AF, ColE1, p15A, pSC101, 2-log ladder. • Since gel verification was successful, performed PCR in triplicate of backbones and origins with same reaction conditions. • Performed PCR cleanup and quantified using Nanodrop spectrophotometer. DVL1_AE had a very low DNA concentration compared to negative control → repeat PCR. • Carried out 50µL MfeI restriction digest and cleanup. • Performed ligation of each backbone with each new origin. and transformed onto Kanamycin (for DVL1 backbones) and Ampicillin (for DVL2 backbones) plates with X-Gal and IPTG. Because of the intact LacZ fragment in the plasmid, the presence of IPTG will allow for activation of β-galactosidase, which will react with X-Gal to produce blue-pigmented colonies. • Successful blue colonies grew for DVL1_ColE1, DVL1_p15A, DVL1_pSC101, and DVL2_ColE1. No growth observed for DVL2_p15A or DVL2_pSC101. → Repeat ligation step from purified RD. • Re-transformed DVL2_p15A and DVL2_pSC101 ligations and performed blue-white screening after overnight incubation. Blue colonies grew for DVL2_pSC101, but blue and white colonies grew for DVL2_p15A. This should not be the case, as any plasmid without the LacZ fragment should not have an origin, and therefore should not have replicated. |
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July |
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Week of July 7 | |
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• Miniprepped liquid cultures of blue colonies of DVL1_ColE1, DVL1_p15A, DVL1_pSC101, and DVL2_ColE1. Sent samples for sequencing, including "VF" and "VR" primers to verify presence of LacZ fragment, and appropriate origin primers to verify new origin presence. • Sequence verification was successful for DVL1_ColE1 and DVL2_ColE1 origins. LacZ presence was confirmed in all sequenced samples, but desired origins were not in remaining samples. → perform colony PCR on DVL1_ColE1, DVL1_p15A, DVL1_pSC101, DVL2_ColE1, DVL2_p15A, and DVL2_pSC101 with origin primers to verify problem. • Gel from colony PCR verified the presence of the origin on DVL1_ColE1 and DVL2_ColE1, but the PCR failed on all others. The likely cause is that the original templates of DVL1_AE, DVL2_AF (with their intact high-copy pMB1 origins), and the complete devices containing the lower copy plasmids made it past the PCR and all purifications. The pXcpi device (containing the p15 ori) had Ampicillin resistance but no LacZ fragment, which explains the white colonies on the DVL2_p15A plate. • To relieve this problem, the purified restriction digests were separated by gel electrophoresis, and a Qiagen gel extraction was performed to remove the bands of desired length. The desired origins and backbones were easy to pick out, since the origins vary from 750bp-950bp, distinguishing them in size from their parent constructs. • The gel extraction was purified and a ligation was carried out to make new DVL1_p15A, DVL1_pSC101, DVL2_p15A, and DVL2_pSC101 plasmids. • The ligations were transformed and large numbers of blue cultures grew for all ligations. The liquid cultures were miniprepped and sent for sequencing. |
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