Team:BostonU/Backbones

From 2014.igem.org

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         <th colspan="2" scope="col">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 <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo</a> for more information on our assembly method). The general plan to replace the backbones was formulated, which comprised of:<br><br>1. Removing the backbones without their high-copy origins from their full destination vectors and removing the lower-copy origins from their plasmids.<br>2.Performing a restriction digest on the backbone and origin fragments to have compatible sticky ends.<br>3. Ligating the origins to the destination vectors.<br>4. Transforming into E. Coli, purifying the plasmid DNA, and sequencing for confirmation.<br><br>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 <a href="https://static.igem.org/mediawiki/2014/c/c6/Primer_Design_6-23_BU14.xls">here</a>)<br><br>
         <th colspan="2" scope="col">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 <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo</a> for more information on our assembly method). The general plan to replace the backbones was formulated, which comprised of:<br><br>1. Removing the backbones without their high-copy origins from their full destination vectors and removing the lower-copy origins from their plasmids.<br>2.Performing a restriction digest on the backbone and origin fragments to have compatible sticky ends.<br>3. Ligating the origins to the destination vectors.<br>4. Transforming into E. Coli, purifying the plasmid DNA, and sequencing for confirmation.<br><br>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 <a href="https://static.igem.org/mediawiki/2014/c/c6/Primer_Design_6-23_BU14.xls">here</a>)<br><br>
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• Struck out<br>
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• Struck out devices with low copy origins for PCR on plates with appropriate antibiotic.<br>
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• Learned how to use miniEugene from CIDAR lab members.<br>
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• Prepared liquid cultures, incubated, and miniprepped.<br>
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• Developed plan for next two weeks that would guide through training of essential lab protocols - specifically plate streaking, minipreps, MoClo, sequencing, and colony PCR.<br>
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• Received primers, diluted.
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• Began using miniEugene to design architecture of constructs.<br>
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• Struck out plates with 15 L0 parts, 6 L1 parts, and 2 L2 parts and grew colonies in liquid cultures overnight.<br>
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<th colspan="2" scope="col"><h3>Week of May 19</h3></th>
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<th colspan="2" scope="col"><h3>Week of June 30</h3></th>
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<th colspan="2" scope="col">• Performed minipreps and quantified the DNA concentration of parts using a Nanodrop spectrophotometer.<br>
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• Sent the parts for sequencing, all but four of which were correctly verified using BLAST. Parts not correctly verified were re-submitted for sequencing.<br>
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• Carried out PCR of DVL1_AE, DVL2_AF, ColE1, p15A, and pSC101.<br>
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• Transformed verified parts into E. Coli Bioline cells and performed colony PCR and gel electrophoresis.</th>
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• Ran gel to confirm primer functionality.<br>
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• GEL 1 HERE.<br>
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• Since gel verification was successful, performed PCR in triplicate of backbones and origins with same reaction conditions.<br>
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Revision as of 15:38, 10 July 2014



Lower Copy Backbones

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 for the same reason. 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.

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. Removing the backbones without their high-copy origins from their full destination vectors and removing the lower-copy origins from their 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)

• 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 1 HERE.
• Since gel verification was successful, performed PCR in triplicate of backbones and origins with same reaction conditions.

Week of May 26

• Analyzed gels and picked colonies.
• Performed minipreps on gel-verified strains.
• Made glycerol stocks of confirmed parts.
• Set up and performed MoClo reactions to make 2 new L1 parts.
• Transformed MoClo reactions and grew up over the weekend.

June

Week of June 2

• Picked colonies, miniprepped, quantifies, and sent parts for sequence verification.
• Set up and performed MoClo reaction for 2 new L1 and 2 new L2 parts.
• Transformed reactions and grew up overnight.
• Brainstormed to further define project goals.

Week of June 9

• Participated in SB2 (Synthetic Biology Boston) and IWBDA (Internatonal Workshop on Bio-Design Automation) workshops hosted at BU.

Week of June 16

• Set up parts for FACS experiment.
• Participated in NEGEM meetup.







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