Team:BostonU/Backbones

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         <th scope="col">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. <br><br>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.</th>
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<th scope="col"><img src="https://static.igem.org/mediawiki/2014/7/75/DVL1_AE_BU14.png" width="300" height="300" alt="DVL1AE" style="float:right" style= "margin-left:10px"><br><br><capt><br>Plasmid map of a MoClo Level 1 destination vector with original pMB1 origin of replication, LacZ fragment, and designed primers for backbone extraction.</capt></th>
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<center><img src="https://static.igem.org/mediawiki/2014/9/99/BU14_New_DVs.png" width="70%"></center><br>
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<th colspan="2" scope="col"><br><h2>June</h2></th>
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<h3>Why Lower Copy Count Origins of Replication?</h3>
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The work carried out by previous BU iGEM teams used destination vectors, or plasmid backbones, with high-copy origins of replication. Having only these high-copy plasmids as an option 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 for all plasmids in their devices. Circuit behavior can be desynchronized with the use of a high copy origins for all plasmids, as it causes over-expression of the plasmid in a cell, leading to a high amount of transcription and protein expression, which can sometimes be toxic or problematic for complex device function. For more complex circuits, this over-expression pushes the limit of the amount of ribosomes that can be sequestered for translation, in addition to straining the cell's protein degradation mechanisms.
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<br><br> Published complex genetic circuits, for example the NOR gates [1] and the Collins counter [2], are often in cloned in medium or low copy vectors, using the ColE1 or p15A origin of replication, respectively. The plasmid maps shown below are from those papers and show the origins of replication used for their work. <br><br>
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<th colspan="2" scope="col"><h3>Week of June 23</h3></th>
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<center><img src="https://static.igem.org/mediawiki/2014/e/e4/BU14_ColE1_p15A.png" width="75%"></center>  
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<capt><center>The image above shows two plasmid maps from the NOR paper [1] (right and middle) and one of the counters [2] (left). These were obtained from the Supplemental Information for both publications.</center></capt>
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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>All protocols used in this notebook are found in our <a href="https://2014.igem.org/Team:BostonU/Protocols">protocols</a> section.<br><br>
 
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• Struck out devices with low copy origins for PCR on plates with appropriate antibiotic.<br>
 
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• Prepared liquid cultures, incubated, and miniprepped.<br>
 
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• Received primers, diluted.
 
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<th colspan="2" scope="col"><br><h3>Week of June 30</h3></th>
 
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<br><br> Detailed progress on new vector backbone creation can be found in the <a href="https://2014.igem.org/Team:BostonU/BackbonesNotebook">backbones notebook</a>.</td></tr>
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• Carried out PCR of DVL1_AE, DVL2_AF, ColE1, p15A, and pSC101.<br>
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<h3>Design and Assembly</h3></td></tr>
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• Ran gel to confirm primer functionality.
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<td scope="col" colspan="2">The origins we selected were sourced from plasmids employed in Prof. Chris Voigt's work on layered logic gates [3]. We selected the ColE1, p15A, and pSC101 origins because they are commonly used origins of replication in the literature for high, medium, and low copy number respectively. The cloning strategy involved using PCR to amplify out the desired backbone from our library without its high copy origin, in addition to the desired origin from its parent construct. The PCR primer sequences added restriction endonuclease sites on each end of the amplicons, one for the enzyme <a href="https://www.neb.com/products/r0589-mfei" target=_blank>MfeI</a> and the other for <a href="https://www.neb.com/products/r0558-asci" target=_blank>AscI</a> These were selected because neither the backbone nor any of the origins contained restriction sites for these endonucleases. The amplicons were digested with both enzymes, and were ligated and transformed.<br></td></tr>
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<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Backbones_Diagram_AscI.png" width="1200px"><br><br><capt><br>Schematic of origin cloning - The PCR primer design added restriction sites for the MfeI and AscI restriction enzymes, 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>).</capt></center></td></tr>
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<h3>Testing</h3>Flow cytometry testing of new low copy backbones has been carried out for the p15A origin against the pMB1 origin, using the <a href="https://2014.igem.org/Team:BostonU/Software#flow">flow cytometry</a> controls. <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo</a> was used to replace the <i>LacZ</i> fragment of the new backbone with a transcriptional unit constitutively expressing fluorescence - all the combinations of fluorescent proteins and origins of replications are found below. More information on the flow cytometry experiments for the backbones can be found in the <a href="https://2014.igem.org/Team:BostonU/BackbonesNotebook">backbones notebook</a>.<br><br><center><img src="https://static.igem.org/mediawiki/2014/1/18/Flow108controls_BU14.png" ><br>
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<capt>The combinations of plasmid origins of replication and fluorescent reporters used for testing the function of the p15A origin of replication against the high-copy pMB1 origin.</capt></center>
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<br><br><img src="https://static.igem.org/mediawiki/2014/2/26/Gel_6-30_BU14.jpg" width="600" height="250" alt="Gel_6-30" style="float:center"><br><capt>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.</capt><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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• Performed PCR cleanup and quantified using Nanodrop spectrophotometer. DVL1_AE had a very low DNA concentration compared to negative control → repeat PCR.<br>
 
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• Carried out 50µL MfeI restriction digest and cleanup.<br>
 
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• 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.
 
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• 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.
 
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• 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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<h3>References</h3><br>
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<th colspan="2" scope="col"><br><h2>July</h2></th>
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[1] A. Tamsir, J. Tabor, C. Voigt (2011). “Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’.” Nature 469: 212-215
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[2] A. Friedland, T. Lu, X. Wang, D. Shi, G. Church, J. Collins (2009). "Synthetic Gene Networks That Count." Science 324:1199-1202 DOI: 10.1126/science.1172005<br>
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[3]  T. S. Moon, C. Lou, A. Tamsir, B. C. Stanton, C. A. Voigt (2012). "Genetic programs constructed from layered logic gates in single cells" Nature 491, 249.
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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.<br>
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• 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.<br>
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• 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.<br>
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• 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.<br>
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• The gel extraction was purified and a ligation was carried out to make new DVL1_p15A, DVL1_pSC101, DVL2_p15A, and DVL2_pSC101 plasmids.
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• The ligations were transformed and large numbers of blue cultures grew for all ligations. The liquid cultures were miniprepped and sent for sequencing.</th>
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Latest revision as of 02:16, 18 October 2014



Lower Copy Backbones


Why Lower Copy Count Origins of Replication?

The work carried out by previous BU iGEM teams used destination vectors, or plasmid backbones, with high-copy origins of replication. Having only these high-copy plasmids as an option 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 for all plasmids in their devices. Circuit behavior can be desynchronized with the use of a high copy origins for all plasmids, as it causes over-expression of the plasmid in a cell, leading to a high amount of transcription and protein expression, which can sometimes be toxic or problematic for complex device function. For more complex circuits, this over-expression pushes the limit of the amount of ribosomes that can be sequestered for translation, in addition to straining the cell's protein degradation mechanisms.

Published complex genetic circuits, for example the NOR gates [1] and the Collins counter [2], are often in cloned in medium or low copy vectors, using the ColE1 or p15A origin of replication, respectively. The plasmid maps shown below are from those papers and show the origins of replication used for their work.

The image above shows two plasmid maps from the NOR paper [1] (right and middle) and one of the counters [2] (left). These were obtained from the Supplemental Information for both publications.


Detailed progress on new vector backbone creation can be found in the backbones notebook.

Design and Assembly

The origins we selected were sourced from plasmids employed in Prof. Chris Voigt's work on layered logic gates [3]. We selected the ColE1, p15A, and pSC101 origins because they are commonly used origins of replication in the literature for high, medium, and low copy number respectively. The cloning strategy involved using PCR to amplify out the desired backbone from our library without its high copy origin, in addition to the desired origin from its parent construct. The PCR primer sequences added restriction endonuclease sites on each end of the amplicons, one for the enzyme MfeI and the other for AscI These were selected because neither the backbone nor any of the origins contained restriction sites for these endonucleases. The amplicons were digested with both enzymes, and were ligated and transformed.



Schematic of origin cloning - The PCR primer design added restriction sites for the MfeI and AscI restriction enzymes, which would give the ends of each of the amplified fragments compatible 4bp overhangs suitable for ligation. (Detailed primer design available here).


Testing

Flow cytometry testing of new low copy backbones has been carried out for the p15A origin against the pMB1 origin, using the flow cytometry controls. MoClo was used to replace the LacZ fragment of the new backbone with a transcriptional unit constitutively expressing fluorescence - all the combinations of fluorescent proteins and origins of replications are found below. More information on the flow cytometry experiments for the backbones can be found in the backbones notebook.


The combinations of plasmid origins of replication and fluorescent reporters used for testing the function of the p15A origin of replication against the high-copy pMB1 origin.

References


[1] A. Tamsir, J. Tabor, C. Voigt (2011). “Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’.” Nature 469: 212-215
[2] A. Friedland, T. Lu, X. Wang, D. Shi, G. Church, J. Collins (2009). "Synthetic Gene Networks That Count." Science 324:1199-1202 DOI: 10.1126/science.1172005
[3] T. S. Moon, C. Lou, A. Tamsir, B. C. Stanton, C. A. Voigt (2012). "Genetic programs constructed from layered logic gates in single cells" Nature 491, 249.







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