Week 12 Presentation | Heejo Choi - Amazon S3€¦ · Week 12 Presentation | Heejo Choi.pptx...
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The case for decoupling assembly and submission standards
to maintain a more flexible registry of biological parts
RN Alnahhas, B Slater, Y Huang, C Mortensen, JW Monk, Y Okasheh, MD Howard, NR GoIel, MJ Hammerling, and JE Barrick
Presented by Heejo Choi Week 12 | 2015 April 29
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BioBrick’s Request for Comments (RFC) Process hosts discussions for the development of standards
• BioBricks Founda=on manages the Registry of Standard Biological Parts o Submission standards – requirements on DNA sequence o Assembly standards – procedure to combine biological parts
• RFCs communicate requests for new standards o Propose a standard o Describe protocols o Comment, extend, or replace previous RFC
• 100+ RFCs documented today
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Current Standard: RFC 10 cons=tutes a combined assembly and submission standard
• Parts must be flanked by prefix and suffix sequences containing restric=on enzyme sites for assembly
• These illegal restric=on sites must not be present within the sequence
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Recent trends suggest a growing number of unreleased parts due to an illegal site
• Data represents parts submiRed by July 29, 2013
• “suggest a significant and growing burden” to adhere RFC 10
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Recent trends weakly suggest a growing number of unreleased parts due to an illegal site
Data represents parts submiRed by July 29, 2013 Adapted from Figure 1C
0
200
400
600
800
1000
2002 2004 2006 2008 2010 2012 2014 Year
Number of unreleased parts with an illegal site
RFC 10
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RFC 95 may alleviate the burden of removing illegal restric=on enzyme sites prior to part submission
• ~1850 par=cipants (iGEM 2013 Annual Review)
• < 3% survey par=cipa=on
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RFC 95 proposes a submission standard decoupled from assembly
• Homing endonucleases recognize (and cleave) longer target sequences o Less likely to find illegal sites within the Part
• RFC 95 can be backwards compa=ble with RFC 10 o Flanking current restric=on enzyme sites with homing endonuclease sites
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Assump=ons
The group’s claims rest on the following assump,ons:
• Field is currently limited by the number of available parts o Perhaps expanding number of available parts is not the limi=ng factor o True challenge is defining the quality, characteriza=on, and documenta=on of parts (Kwok 2010)
• Transi=on from RFC 10 to RFC 95 is larger than implied
o RFC 95 is not always backwards-‐compa=ble with RFC 10 o Illegal sites within the part must s=ll be removed o Majority of teams use restric=on enzyme cloning methods
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Concerns and Sugges=ons
Concerns: • Survey data rests on < 3% response • Paper may be ahead of its =me (field is not limited by the number of available parts)
Sugges,ons:
• Acquire more data points for survey (Figure 2) • Clarity of how data in Figure 1C • Consider mandatory ques=onnaire for a representa=ve random pool • Ask direct ques=ons:
o “Would your team prefer to use RFC 95 as the standard rather than RFC 10?” o “Does your team feel the Registry needs more standard parts?”
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Significance of data cura=on & informa=on accessibility
• Registry of Standard Biological Parts is ever growing o Philosophy of “Get, Give & Share”
• Standardiza=on ensures compa=bility of parts when crea=ng longer, more complex parts
• Cura=on process is limited by quality part characteriza=on
o Exemplified by diverse degree of detail for logic gate entries in SynBioLGDB
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Significance of an=cipa=ng change
“For a gene=c parts repository and registry to remain relevant as technology progresses, it should an=cipate these changes and adapt its methods to complement them.” Future Work • RFC 98 (rapid assembly of RFC 10 compa=ble parts in a single reac=on)
• RFC 104 (rapid assembly of RFC 10 parts without using restric=on enzymes)
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The Backstory from M. Hammerling (2012 UT Aus=n iGEM Team)
• SubmiRed parts were rejected due to illegal sites • “While we made it to the interna=onal compe==on at MIT that year, the team was
unwilling to completely re-‐engineer a perfectly func=onal and well-‐characterized biological part to comply with what we viewed as an arbitrary sequence constraint. This limited how much success we were able to achieve at the interna=onal level that year. ”
• “…introduce a new submission standard that would fix this problem and allow
teams to focus on developing novel, useful parts rather than muta=ng the sequences of their parts to comply with the current submission standard.”
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U=lizing homing endonuclease sites reduces the probability of encountering a illegal site within part
• Restric=on enzyme sites (6-‐8 bp)
• Homing endonuclease sites (15-‐30 bp)
Most used rank Part Length
(bp)
P(illegal site in part)
1 RBS 12 2-‐3%
2 Terminator 129 4-‐12%
9 GFP 720 50%
from Top 10 Most Used Parts on the Registry (hRp://parts.igem.org/Frequently_Used_Parts)