schematic of integrase-based continuous event detector

SURF 2018: Integrase-based genetic circuits

2017-12-14T01:41:22Z

Ashur:

'''[[SURF 2018|2018 SURF]] project description'''
* Mentor: Richard Murray
* Co-mentor: Andrey Shur

Integrases are bacteriophage proteins that catalyze recombination of phage DNA with the bacterial genome at specific DNA sequences known as attachment (att) sites. Serine integrases in particular catalyze directional recombination between cognate att sites without additional cofactors and therefore are valuable tools in synthetic biology. Previous work has utilized serine integrases and attachment sites as black boxes that inducibly flip or excise DNA to construct genetic memory, logic gates, and event detectors. We recognize that the ability to induce DNA changes can be a powerful tool in synthetic biology, since DNA is an easily tractable medium for information storage and of course encodes all the proteins and genes needed for life. Thus we are interested in expanding the list of different "functions" that integrases can perform as well as thinking about new and interesting ways to utilize the functions that are already known. A few descriptions of ongoing projects in this area are below.

'''Integrase-based continuous event logging'''

Biological records of events are omnipresent in paleontology, history, and climate science. Tree rings and ice cores provide evidence of environmental conditions that have been recorded in the composition of living cells that are deposited over time, carrying with them the a record of events that have influenced their lives before being buried underneath ice or inside the trunk of a tree. Using integrases, customizable biological memory devices can be made to record any event that a bacterium can detect. Bacteria can be unobtrusively seeded in a natural environment, providing a stable genetic record on any timescale that is suitable to the researcher.

'''Field programmable circuits in bacteria'''

Microchips with a specific purpose are expensive and time consuming to make. For many applications, it is sufficient to make a chip containing a set of unconnected logic gates and other elements, and allow the user to define the connections between these components after the chip has already been made. This concept is known as a programmable interconnect. Using integrases it is possible to develop a programmable interconnect in bacteria. A set of circuits and useful components can be integrated in the bacterial genome in a non-functional form. This way, the genes are not impacting the health of the bacterium if they are not needed. A set of integrase operations can then be used to selectively enable and connect disparate circuit elements in vivo, without the need for traditional mo-lecular cloning. This could allow one strain to have many different uses and functions without compromising the viability and stability of its genome. Users would simply program the strain before use, removing or not activating unneeded circuit components at will.

'''Prerequisite Skills'''
* Molecular cloning
** Golden Gate assembly
** Gibson assembly
** Restriction cloning
** Primer design
** Bacteria culture
** Miniprep
* Python

'''References'''
# Bonnet, J., Subsoontorn, P. & Endy, D. [http://www.pnas.org/content/109/23/8884.full Rewritable digital data storage in live cells via engineered control of recombination directionality]. Proc. Natl. Acad. Sci. 109, 8884–8889 (2012).
# Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S. & Lu, T. K. [http://science.sciencemag.org/content/353/6297/aad8559.full Synthetic recombinase-based state machines in living cells]. Science (80-. ). 353, aad8559-aad8559 (2016).
# Hsiao, V., Hori, Y., Rothemund, P. W. K. & Murray, R. M. [http://onlinelibrary.wiley.com/doi/10.15252/msb.20156663/full A population-based temporal logic gate for timing and recording chemical events]. Mol. Syst. Biol. 557, 1–17 (2016).
# Smith, M. C. A., Till, R. & Smith, M. C. M. [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2003.03942.x/full Switching the polarity of a bacteriophage integration system]. Mol. Microbiol. 51, 1719–1728 (2004).
# Shur, A. & Murray, R. M. [https://www.biorxiv.org/content/early/2017/02/21/110254 Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli]. bioRxiv (2017). doi:10.1101/110254
# Shur, A. & Murray, R. M. [https://www.biorxiv.org/content/biorxiv/early/2017/11/25/225151.full.pdf Proof of concept continuous event logging in living cells]. bioRxiv (2017). doi:10.1101/225151

2016-06-22T02:24:01Z

Ashur: /* CIDAR */

Moclo is a type of cloning strategy that attempts to standardize the assembly of multiple DNA parts into finished constructs or 'devices'. The basic cloning method relies on the fact that typeIIs restriction enzymes cut outside their sequence of recognition. Unlike traditional restriction cloning, this means there is no chance that ligated pieces can be cut again by the same enzyme. This also gives you complete freedom in choosing the four base-pair sticky end, and allows multiple specific ligations to be accomplished with only one restriction enzyme.

In order to take full advantage of the modularity and ease of use, one must spend significant time "domesticating" DNA parts by removing extra restriction sites and placing them into appropriate vectors which contain the typeIIs restriction sites in the right orientation. This work has been done by many labs (with equally many sticky end overhang standards).

One example of such a standard is the CIDAR library from Doug Densmore's lab

=CIDAR=
[[File:CIDAR.png|right|500px]]
{| class="wikitable sortable"
! Name !! Well !! Type !! Overhangs !! Description !! Link
|-
| J23100_AB || A1 || Promoter || AB || Weak Constitutive Promoter || https://benchling.com/s/gS6lXC3X/edit
|-
| J23100_EB || A2 || Promoter || EB || Weak Constitutive Promoter || https://benchling.com/s/aq8Gxp2O/edit
|-
| J23100_FB || A3 || Promoter || FB || Weak Constitutive Promoter || https://benchling.com/s/rTzq9ToN/edit
|-
| J23100_GB || A4 || Promoter || GB || Weak Constitutive Promoter || https://benchling.com/s/5yn5Zy6g/edit
|-
| J23102_AB || A5 || Promoter || AB || Strong Constitutive Promoter || https://benchling.com/s/aQOLd1xr/edit
|-
| J23102_EB || A6 || Promoter || EB || Strong Constitutive Promoter || https://benchling.com/s/zOCQC6dM/edit
|-
| J23102_FB || A7 || Promoter || FB || Strong Constitutive Promoter || https://benchling.com/s/1pfiY4NG/edit
|-
| J23102_GB || A8 || Promoter || GB || Strong Constitutive Promoter || https://benchling.com/s/LmBWThyt/edit
|-
| J23103_AB || A9 || Promoter || AB || Weak Constitutive Promoter || https://benchling.com/s/9KEsWqta/edit
|-
| J23103_EB || A10 || Promoter || EB || Weak Constitutive Promoter || https://benchling.com/s/ynovZBP9/edit
|-
| J23103_FB || A11 || Promoter || FB || Weak Constitutive Promoter || https://benchling.com/s/oZSwbWfi/edit
|-
| J23103_GB || A12 || Promoter || GB || Weak Constitutive Promoter || https://benchling.com/s/4qwyO9ja/edit
|-
| J23106_AB || B1 || Promoter || AB || Medium Constitutive Promoter || https://benchling.com/s/riO8LQFW/edit
|-
| J23106_EB || B2 || Promoter || EB || Medium Constitutive Promoter || https://benchling.com/s/ljtaz48p/edit
|-
| J23106_FB || B3 || Promoter || FB || Medium Constitutive Promoter || https://benchling.com/s/b8wazW5M/edit
|-
| J23106_GB || B4 || Promoter || GB || Medium Constitutive Promoter || https://benchling.com/s/UqDZklx7/edit
|-
| J23107_AB || B5 || Promoter || AB || Medium Constitutive Promoter || https://benchling.com/s/G8ouTflM/edit
|-
| J23107_EB || B6 || Promoter || EB || Medium Constitutive Promoter || https://benchling.com/s/SGzlA9lZ/edit
|-
| J23107_FB || B7 || Promoter || FB || Medium Constitutive Promoter || https://benchling.com/s/0mSZV0LM/edit
|-
| J23107_GB || B8 || Promoter || GB || Medium Constitutive Promoter || https://benchling.com/s/InUihbMF/edit
|-
| J23116_AB || B9 || Promoter || AB || Weak Constitutive Promoter || https://benchling.com/s/1mQPZFtM/edit
|-
| J23116_EB || B10 || Promoter || EB || Weak Constitutive Promoter || https://benchling.com/s/xqAO8tJl/edit
|-
| J23116_FB || B11 || Promoter || FB || Weak Constitutive Promoter || https://benchling.com/s/o8lvrIdJ/edit
|-
| J23116_GB || B12 || Promoter || GB || Weak Constitutive Promoter || https://benchling.com/s/YYNkGLBV/edit
|-
| R0010_AB || C1 || Promoter || AB || pLac LacI Repressible Promoter || https://benchling.com/s/etg7OrN6/edit
|-
| R0010_EB || C2 || Promoter || EB || pLac LacI Repressible Promoter || https://benchling.com/s/FamBps3h/edit
|-
| R0010_FB || C3 || Promoter || FB || pLac LacI Repressible Promoter || https://benchling.com/s/oi1T00L0/edit
|-
| R0010_GB || C4 || Promoter || GB || pLac LacI Repressible Promoter || https://benchling.com/s/Yw6bgPjg/edit
|-
| R0040_AB || C5 || Promoter || AB || pTet Strong TetR Repressible Promoter || https://benchling.com/s/3RYAjD6u/edit
|-
| R0040_EB || C6 || Promoter || EB || pTet Strong TetR Repressible Promoter || https://benchling.com/s/SNZHRImR/edit
|-
| R0040_FB || C7 || Promoter || FB || pTet Strong TetR Repressible Promoter || https://benchling.com/s/nyzVqekN/edit
|-
| R0040_GB || C8 || Promoter || GB || pTet Strong TetR Repressible Promoter || https://benchling.com/s/473M59P8/edit
|-
| R0063_AB || C9 || Promoter || AB || pLux Weak LuxR Repressible Promoter || https://benchling.com/s/EUFuZKHP/edit
|-
| R0063_EB || C10 || Promoter || EB || pLux Weak LuxR Repressible Promoter || https://benchling.com/s/PN1VmcWg/edit
|-
| R0063_FB || C11 || Promoter || FB || pLux Weak LuxR Repressible Promoter || https://benchling.com/s/xnA98Y5U/edit
|-
| R0063_GB || C12 || Promoter || GB || pLux Weak LuxR Repressible Promoter || https://benchling.com/s/jHQHwAaD/edit
|-
| I13453_AB || D1 || Promoter || AB || pBAD AraC Inducible Promoter || https://benchling.com/s/HE4joK6k/edit
|-
| I13453_EB || D2 || Promoter || EB || pBAD AraC Inducible Promoter || https://benchling.com/s/YC8NeyAe/edit
|-
| I13453_FB || D3 || Promoter || FB || pBAD AraC Inducible Promoter || https://benchling.com/s/c4zFwYww/edit
|-
| I13453_GB || D4 || Promoter || GB || pBAD AraC Inducible Promoter || https://benchling.com/s/i9YCgIRi/edit
|-
| B0032m_BC || D5 || RBS || BC || Weiss Medium Strength RBS || https://benchling.com/s/jwIsLDg9/edit
|-
| B0033m_BC || D6 || RBS || BC || Weiss Low strength RBS || https://benchling.com/s/FQFaG78R/edit
|-
| B0034m_BC || D7 || RBS || BC || Weiss High Strength RBS || https://benchling.com/s/kBNYDAHo/edit
|-
| BCD12_BC || D8 || RBS || BC || Medium Strength BCD RBS || https://benchling.com/s/mUcZUqJo/edit
|-
| BCD2_BC || D9 || RBS || BC || High Strength BCD RBS || https://benchling.com/s/jM8sZdRg/edit
|-
| BCD8_BC || D10 || RBS || BC || Low Strength BCD RBS || https://benchling.com/s/8UhD5Vaf/edit
|-
| C0012m_CD || D11 || CDS || CD || LacI Repressor protein || https://benchling.com/s/1RopAYaI/edit
|-
| C0040_CD || D12 || CDS || CD || TetR Repressor Protein || https://benchling.com/s/M9uZpTOg/edit
|-
| C0062_CD || E1 || CDS || CD || LuxR Repressor/Activator Protein || https://benchling.com/s/FFyxWiwT/edit
|-
| C0080_CD || E2 || CDS || CD || LuxR Repressor/Activator Protein || https://benchling.com/s/FFyxWiwT/edit
|-
| cre_CD || E3 || CDS || CD || Cre Recombinase Protein || https://benchling.com/s/CAXPcaMy/edit
|-
| E0030_CD || E4 || CDS || CD || Yellow Fluorescent Protein || https://benchling.com/s/cr12BVOE/edit
|-
| E0040m_CD || E5 || CDS || CD || Green Fluorescent Protein || https://benchling.com/s/cj91KrVH/edit
|-
| E1010m_CD || E6 || CDS || CD || Red Fluorescent Protein || https://benchling.com/s/1AbCv00F/edit
|-
| eBFP2_CD || E7 || CDS || CD || Blue Fluorescent Protein || https://benchling.com/s/bbkctFdr/edit
|-
| B0015_DE || E8 || Terminator || DE || Double Terminator || https://benchling.com/s/a1cHND33/edit
|-
| B0015_DF || E9 || Terminator || DF || Double Terminator || https://benchling.com/s/f6RaEG2K/edit
|-
| B0015_DG || E10 || Terminator || DG || Double Terminator || https://benchling.com/s/5pFYTkSF/edit
|-
| B0015_DH || E11 || Terminator || DH || Double Terminator || https://benchling.com/s/8st8vLlQ/edit
|-
| DVA_AB || E12 || Destination || AB || Moclo Part Destination Vector (stage 1), Promoter || https://benchling.com/s/slHkWK6p/edit
|-
| DVA_AE || F1 || Destination || AE || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/JbqtviMA/edit
|-
| DVA_AF || F2 || Destination || AF || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/NviiNLIp/edit
|-
| DVA_AG || F3 || Destination || AG || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/pPe92CO5/edit
|-
| DVA_AH || F4 || Destination || AH || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/iaYDvlV2/edit
|-
| DVA_BC || F5 || Destination || BC || Moclo Part Destination Vector (stage 1), UTR || https://benchling.com/s/o7TOrROp/edit
|-
| DVA_CD || F6 || Destination || CD || Moclo Part Destination Vector (stage 1), CDS || https://benchling.com/s/xgdB8Znv/edit
|-
| DVA_DE || F7 || Destination || DE || Moclo Part Destination Vector (stage 1), Terminator || https://benchling.com/s/mtpMynxT/edit
|-
| DVA_DF || F8 || Destination || DF || Moclo Part Destination Vector (stage 1), Terminator || https://benchling.com/s/53Whxb2s/edit
|-
| DVA_DG || F9 || Destination || DG || Moclo Part Destination Vector (stage 1), Terminator || https://benchling.com/s/bJdQOwZP/edit
|-
| DVA_DH || F10 || Destination || DH || Moclo Part Destination Vector (stage 1), Terminator || https://benchling.com/s/sGGQDAg6/edit
|-
| DVA_EB || F11 || Destination || EB || Moclo Part Destination Vector (stage 1), Promoter || https://benchling.com/s/i5JEIhUg/edit
|-
| DVA_EF || F12 || Destination || EF || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/OlSBaZCj/edit
|-
| DVA_EG || G1 || Destination || EG || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/xGeYtbZx/edit
|-
| DVA_EH || G2 || Destination || EH || Moclo Device Destination Vector (stage 3) || https://benchling.com/s/fnTqha2S/edit
|-
| DVA_FB || G3 || Destination || FB || Moclo Part Destination Vector (stage 1), Promoter || https://benchling.com/s/3CuYwFwU/edit
|-
| DVA_GB || G4 || Destination || GB || Moclo Part Destination Vector (stage 1), Promoter || https://benchling.com/s/UvuVapbV/edit
|-
| DVA || G5 || Vector || - || Vector for new Stage 1 or 3 vector || https://benchling.com/s/6hijqfvT/edit
|-
| pJ02B2Gm_AE || G6 || Device || AE || High GFP Expression Device, in Part Vector || https://benchling.com/s/mgYDcL4z/edit
|-
| pJ02B2Gm_EF || G7 || Device || EF || High GFP Expression Device, in Part Vector || https://benchling.com/s/3WQbdlzU/edit
|-
| pJ02B2Rm_AE || G8 || Device || AE || High RFP Expression Device, in Part Vector || https://benchling.com/s/Kb4AAbox/edit
|-
| pJ02B2Rm_EF || G9 || Device || EF || High RFP Expression Device, in Part Vector || https://benchling.com/s/FOM8uxtS/edit
|-
| pJ02B2Gm:Rm_AF || G10 || Device || AF || High GFP and High RFP expression Device, in Part Vector || https://benchling.com/s/D75ziv6K/edit
|-
| pJ02B2Rm:Gm_AF || G11 || Device || AF || High RFP and High GFP Expression Device, in Part Vector || https://benchling.com/s/OOkaVw1a/edit
|-
| pJ02B2Gm_AE || G12 || Device || AE || High GFP Expression Device, in Stage 2 Vector || https://benchling.com/s/ShVJxDdF/edit
|-
| pJ02B2Gm_EF || H1 || Device || EF || High GFP Expression Device, in Stage 2 Vector || https://benchling.com/s/uUTmefO7/edit
|-
| pJ02B2Rm_AE || H2 || Device || AE || High RFP Expression Device, in Stage 2 Vector || https://benchling.com/s/syrZcMB4/edit
|-
| pJ02B2Rm_EF || H3 || Device || EF || High RFP Expression Device, in Stage 2 Vector || https://benchling.com/s/nNICVGuk/edit
|-
| DVK_AE || H4 || Destination || AE || Moclo Device Destination Vector (stage 2) || https://benchling.com/s/sueyphOw/edit
|-
| DVK_AF || H5 || Destination || AF || Moclo Device Destination Vector (stage 2) || https://benchling.com/s/1i0mX41y/edit
|-
| DVK_EF || H6 || Destination || EF || Moclo Device Destination Vector (stage 2) || https://benchling.com/s/7FuJO5tD/edit
|-
| DVK_FG || H7 || Destination || FG || Moclo Device Destination Vector (stage 2) || https://benchling.com/s/Cz5hQ9wM/edit
|-
| DVK_GH || H8 || Destination || GH || Moclo Device Destination Vector (stage 2) || https://benchling.com/s/MFw9pNDU/edit
|-
| DVK || H9 || Vector || - || Vector for new Stage 2 vector || https://benchling.com/s/olS42cDR/edit
|}

2016-06-22T01:32:22Z

Ashur:

Moclo

2016-06-22T01:26:39Z

Ashur: Created page with "Moclo is a type of cloning strategy that attempts to standardize the assembly of multiple DNA parts into finished constructs or 'devices'. The basic cloning method relies on t..."

Research meetings, Jan/Feb 2016

2016-01-18T21:47:18Z

Ashur: /* 26 Jan 2016 (Tue) */

Please sign up for a slot below. __NOTOC__

{| border=1 width=100%
|- valign = top
|
=== 25 Jan 2016 (Mon) ===
* Richard in SF
|
=== 26 Jan 2016 (Tue) ===
* 1-2 pm: Andrey Shur
* 2-3 pm: Anders Knight
* 3-4 pm: Reed McCardell
* 5:30-6:30 pm: Ania Baetica
|

=== 27 Jan 2016 (Wed) ===
* 8:30-9:30 am: open
* 9:30-10:30 am: open
* 4-5 pm: open
* 5-6 pm: open
|
=== 28 Jan 2016 (Thu) ===
* 5:30-6:30 pm: open
* 6:30-7:30 pm: open
|
=== 29 Jan 2016 (Fri) ===
* 2-3 pm: open
* 3-4 pm: open
* 4:30-5:30 pm: open
* 5:30-6:30 pm: open
|- valign = top
|
=== 25 Jan 2016 (Mon) ===
* Richard in Hartford
|
=== 2 Feb 2016 (Tue) ===
* 9-10 am: open
* 2-3 pm: open
* 3-4 pm: open
* 4-5 pm: open
* 5:30-6:30 pm: open
* 6:30-7:30 pm: open
|
=== 3 Feb 2016 (Wed) ===
* 8:30-9:30 am: open
* 9:30-10:30 am: open
|
=== 4 Feb 2016 (Thu) ===
* Richard in SF
|
=== 5 Feb 2016 (Fri) ===
* BE visiting day
|}

BE 150/Bi 250 Spring 2014, Project presentation schedule

2014-05-21T05:10:56Z

Ashur: /* Monday, 1 June 2014 */

Attached below is the schedule for project presentation for BE 150/Bi 250, Spring 2014. Each team should sign up for one 30 minute slot. Please list a tentative title for your talk and the names or initials of the group members who will present. You should prepare a 20 minute talk and then we'll have ~10 minutes of Q&A.

'''Please leave at least one slot in the middle or at the end of each session unfilled, so that we have some buffer time (just in case).'''

=== Thursday, 29 May 2014 ===
* 2:00 pm: Open
* 2:30 pm: Open
* 3:00 pm: Open
* 3:30 pm: Open (use only if no other slots are available)
* 4:00 pm: Open
* 4:30 pm: lncRNA toggle switch (Chris, Tobin, Sofi)

=== Friday, 30 May 2014 ===
* 10:00 am: Open
* 10:30 am: Open
* 11:00 am: C.Elegans Defecation Cycle (Jeff)
* 11:30 am: NADPH to Apoptosis (Ted, Zach, and Dennis)

=== Monday, 2 June 2014 ===
* 2:00 pm: Reaction-diffusion models: Claire and Travis
* 2:30 pm: third time is the charm: Stone Alex and Kevin
* 3:00 pm: Actin regulation, Andrey, Andrew
* 3:30 pm: Open (use only if no other slots are available)
* 4:00 pm: Predator-Prey Model, Julia, Christopher, Belen
* 4:30 pm: Coupled Oscillators with Delays - Harry & Noah

BE 150/Bi 250 Spring 2014, Project presentation schedule

2014-05-20T17:13:20Z

Ashur: /* Monday, 1 June 2014 */

Attached below is the schedule for project presentation for BE 150/Bi 250, Spring 2014. Each team should sign up for one 30 minute slot. Please list a tentative title for your talk and the names or initials of the group members who will present. You should prepare a 20 minute talk and then we'll have ~10 minutes of Q&A.

'''Please leave at least one slot in the middle or at the end of each session unfilled, so that we have some buffer time (just in case).'''

=== Thursday, 29 May 2014 ===
* 2:00 pm: Open
* 2:30 pm: Open
* 3:00 pm: Open
* 3:30 pm: Open (use only if no other slots are available)
* 4:00 pm: Open
* 4:30 pm: lncRNA toggle switch (Chris, Tobin, Sofi)

=== Friday, 30 May 2014 ===
* 10:00 am: Open
* 10:30 am: Open
* 11:00 am: Open
* 11:30 am: Open (use only if no other slots are available)

=== Monday, 1 June 2014 ===
* 2:00 pm: Open
* 2:30 pm: Open
* 3:00 pm: Actin regulation, Andrey, Andrew
* 3:30 pm: Open (use only if no other slots are available)
* 4:00 pm: Predator-Prey Model, Julia, Christopher, Belen
* 4:30 pm: Coupled Oscillators with Delays - Harry & Noah