Team:BostonU/BackbonesNotebook

From 2014.igem.org

• TEAM
• PROJECT
  • Overview
  • Priority Encoder
  • MoClo Assembly Method
  • Low Copy Backbones
  • Tandem Promoters
  • Fusion Proteins
  • Repressor Proteins
  • Multiplexing
  • BioDesign Automation Tools
  • Future Work
• ACHIEVEMENTS
  • Data Collected
  • Parts Submitted
  • Chimera Workflow
  • Chimera Example
  • Medal Fulfillment
• NOTEBOOK
  • Training
  • Protocols
  • Backbones
  • Tandem Promoters and Repressors
  • Fusion Proteins
• CONSIDERATIONS
  • Collaborations
  • Measurement Track
  • Safety
  • Interlab Study
  • NEGEM
  • Policy and Practices
• ACKNOWLEDGEMENTS

Notebook: Lower Copy Backbones

This notebook details the process undertaken to replace the high copy pMB1 origin in our existing MoClo 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. All protocols used in this notebook are found in our protocols section.	Plasmid map of a MoClo Level 1 destination vector with original pMB1 origin of replication, LacZ fragment, and designed primers for backbone extraction.
June
Week of June 23
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. (Detailed primer design available here) 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.
• Struck out devices with low copy origins for PCR on plates with appropriate antibiotic. • Prepared liquid cultures, incubated, and miniprepped. • Received primers, diluted.
Week of June 30
• Carried out PCR of DVL1_AE, DVL2_AF, ColE1, p15A, and pSC101. • Ran gel to confirm primer functionality. Gel verifying the successful PCR extraction of the desired backbones (~2kb each) and origins (~750-950kb). 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.
July
Week of July 7
• 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.
Week of July 14
• Ran temperature-gradient PCR from 48˚C to 58˚C to identify optimal annealing temperature for p15A and pSC101 primers. • 54˚C identified as optimal annealing temperature. • Ran restriction digest and ligated overnight (12 hrs) at 16˚C. • Sequencing for p15A and pSC101 still failing
Week of July 21
• A colony PCR on the p15A and pSC101 colonies from last week's transformations was run with their primers to determine if the correct colonies are present. • The colony PCR failed with all p15A and pSC101 samples. • The purified restriction digest products of the origins were sent for sequencing and returned correct results, so the incorrect final product is likely a ligation issue. • A 2:1 insert:backbone ratio ligation was carried out, transformed, prepped and sequenced. The sequencing reactions for p15A and pSC101 failed.
Week of July 28
• A GFP single expression control was stuck out and miniprepped as preparation for a FACS experiment to test the functioning of the ColE1 clones vs the controls' pMB1 origins. • A new PCR with new reagents was carried out for the p15A and pSC101 origins, in addition to DVL1_AE and DVL2_AF backbones, all the results of which were successfully verified in a 1% agarose gel. • A MoClo reaction was carried out to clone a constitutively-expressed GFP transcriptional unit into the DVL1_AE_ColE1 plasmids. • FACS was successful for the control and the ColE1 clone, although their fluorescence measures were the same.

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