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| Our local Maryland Synthetic Biology team came together to explore the techniques and hardware utilized. We've formed our iGEM team to inspire innovation with goal oriented time lines.
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Possible Projects
Project: DIY-Gem
Creating the set of educational tools and learning resources to allow comprehension and accessibility to the core techniques associated with Synthetic Biology. We've discussed creating e.coli that can be added to the bb system that can actually produce the major enzymes used in these techniques so that beginner's can "grow their own", saving the $1 a ul that some of these can cost. We have the sequence for Pol1 (J)
for Taq Aquaticus, and can put it into e. coli, but it contains a Pst1 site dead in the center of it's sequence so we'd have to create a new sequence and test it's viability, prior to formatting it in the bb format. The Pfu polymerase from Pyroclase Fusarium is usually used with BB, as it has enhanced error protection mechanisms and can withstand higher temperature cycling. The patent on Pol1 has expired. Patents exist on Pst1, none on exist on EcoR1, Xbe and Spe, couldn't find patent or sequence information, need more research. Other implementation of the DIY-Bio structure involves the construction of inexpensive hardware that is utilized in the process of synthetic biology research, i.e. microgram scales, centrifuges, PCR thermocyclers, Gel Electrophoresis kits, Devices for volumetrics, histobots, DNA sequencers/synthesizers and enhanced microscopy equipment. Many of these hw pieces have already been hacked by various groups within the DIY-Bio communities, however our goal would be to create those pieces necessary for us to continue to experiment/collaborate past the end of the iGEM timeframe.
Project: Children of Men
Bio-remediation oriented project to sequester/breakdown excessive estrogen levels that are currently responsible for turning the bass populations in Maryland tributaries into intersex species. Apparently 100% of the Male Bass populations are currently intersex meaning that they are carrying eggs due to the high-levels of hormone disruptors such as Estrogen that are not being cleaned by Maryland's current water treatment facilities. Researchers at Washington State have posited using Ammonia based microbials in bio-reactor systems to help break down estrogen, and there are supposedly BB parts that can be used as Estrogen Receptors. Failure to act, could conceivably lead to a sterile future for mankind, aka "The Children of Men" scenario from the science fiction film of same name.
Similar Bio-remediation scenarios were presented to deal with excess Nitrogen fixation, Pfisteria Sensors and petroleum digestion in response to the Gulf tragedy.
Project: ANN
Artificial neural networks offer powerful tools for pattern recognition, discriminant analysis and machine learning. Originally developed as a model of human cognitive activity, artificial neural networks have been adopted by
statistical modelers for their capacity to partition high-dimensional parameter spaces and "learn" to classify inputs through teaching and reinforcement. The massively parallel nature of artificial neural networks have provided motivation to implement them in-vitro rather than in-silico. Indeed, steps toward in vitro implementation have been taken by a number of previous iGEM teams. We hope to improve upon these efforts.
In particular, we wish to provide tools for the construction of a multi-layer feed forward net with back-propagation of error. Owing to the parallel nature of the net, implementation must consist of the development of a single computational unit along with the processes by which units can be rationally composed. Such a project will require the completion of several subtasks.
The first item is the acceptance of input by the user. We intend to employ cellular signaling mechanisms for communicating with the input layer. Between network layers, we intend to employ the addressible conjugation method developed by Berkeley 2006.After input has been received by a node in a given layer of the net, it must be weighted, summed, and fed through a non-linear threshold function. Weighting may be accompolished by molecular AND gates which limit the expression of the input protein to levels which may be governed by the concentrations of "weighting proteins" within the cell. Summing and thresholding may then be naturally accomplished by cellular metabolism. Output may be given to the user through the expression of fluorescent
proteins. Lastly, back-propagation of error may be accomplished by separate channels of addressing plasmids which up- or down-regulate rates of conjugation in the previous layer.
The above constitutes an ambitious program which may exceed the scope of the summer. We wish to focus our efforts upon techniques for parallel, asynchronous addressible conjugation, especially so as to permit input from multiple nodes in the previous layer and allow computation of multiple input instances through a single generation of cells.
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| Team Example
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DIY-GEM: a path towards low cost high throughput gene synthesis.
Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development.
Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler.
Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects."
== PoliColi Project Details==
Thermus Aquaticus Polymerase I
PolI
J04639.1
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128
1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC
51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG
101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC
151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT
201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG
251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC
301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC
351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC
401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC
451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA
501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG
551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG
601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA
651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC
701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG
751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC
801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG
851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA
901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA
951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC
1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG
1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC
1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG
1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC
1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC
1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT
1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG
1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT
1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA
1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC
1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT
1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC
1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC
1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC
1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA
1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC
1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC
1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA
1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC
1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA
2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA
2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG
2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG
2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG
2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC
2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG
2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG
2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC
2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC
2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG
2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG
2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG
2601 GCTCTCCGCC AAGGAGTGAT ACCACC
We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA.
Plasma DNA is freeware from University of Helsinki which provides quick analysis of plasmid sequence information. http://research.med.helsinki.fi/plasmadna/
When we cut and paste this dna sequence into plasmadna and look at the output window, we are given a visual output of various coding information. Such as restriction sites found within the code. To consider a construct viable for a BbPart
we'll need to make certain that the standard restriction enzymes used with the system won't sheer the dna making it incomplete code. Searching for EcoRI, Xbe1, Sbe1, Pst1 sites will show whether the code is viable in an untampered state.
Problem: PstI restriction site - Found @ 1717
CTGCAG-PstI restriction site
GACGTC-Complement
Solution - Site-specific Mutagenesis by Overlap Extension
Sambrook, Joseph; Russell, David W. ; Molecular Cloning: A Laboratory Manual, 3rd Edition - (http://www.cshlpress.com/default.tpl?cart=1279686078181232350&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=21)
We then used the Gene Designer 2.0 freeware from DNA2.0 (https://www.dna20.com/genedesigner2/) - to analyze the Open Reading Frames. It shows us the Amino Acid codons that were being coded within that PstI Restrictions site. We find that the first three are coding for Leucine with CTG and can be changed at one point to CTT and still maintain Leucine's amino acid. The hope is that this will maintain functional integrity in the manufactured enzyme.
Design 2 Primers
(11-14 Bp around chosen mutation) with changed Amino Acid Bp's Targeting initial Leucine at G of CTG to CTT
* - Point mutation Original G in CTG of Leucine. Change of one base to CTT maintains Leucine integrity.
GTGGAGAAGATCCT(T)CAGTACCGGCGG
CACCTCTTCTAGGA(A)GTCATGGCCGCC
And while we're designing primers, besides the point mutation, we'll take the opportunity to design and order the primers for the Bb Suffix and Prefix. We'll follow the examples laid out in the Registry of Standard Parts, under Promoter Construction - 1. Designing the oligos needed to make a part.
http://partsregistry.org/Help:Promoters/Construction
Important considerations are Melting Point and percentage CG complements. Other considerations are dimerizations, that might cause primers to hairpin.
We analyzed these primers using the OligoAnalyzer at IDT. When analyzing PolI Complements only were used for sequence inquiry, not the Bb Suffix/Prefixes.
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
PolI Coli Primers For Overlap Extension PCR
PCR Reaction - 1
Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC
60.5 c ; 56.5 % GC Concetration
TAQ Rm
CTCCCGGTACTGAAGGATCTTCTCCAC
61.5 c ; 55.6 % GC Concentration
PCR Reaction - 2
TAQ Fm
GTGGAGAAGATCCTTCAGTACCGGGAG
61.5 c; 55.6 % GC
Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC
61.8 c; 65 % GC
PCR Reaction - 3
Bb Prefix & Suffix Primers
Resuspend in 100 uL of H2O
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)
NEXT
- Create Full Bb Prmr w Plasmid combining new part using
<partinfo>R0010</partinfo> - Promoter (LacI)
<partinfo>B0034</partinfo> - Strong RBS
NEW PART - PolI Bb Format
<partinfo>B0015</partinfo> - Double Terminator
Psb1_?_3 - Plasmid of Interest with Chosen Resistance : http://partsregistry.org/Plasmid_backbones
<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS
Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI
Cut <partinfo>B0034</partinfo> w/XbeI & PstI
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because
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New Part + <partinfo>B0015</partinfo> = New Part
Cut New Part w/EcoRI & SpeI
Cut <partinfo>B0015</partinfo> w/XbeI & PstI
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)
Cut 1st Combined Part w/EcoRI & SpeI
Cut 2nd Combined Part w/XbeI & PstI
Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)
Voila!!! Brand New Taq Polymerase Bb Part.
Part 2
The Experiments
We began the first few weeks meeting to orient ourselves with the structure of the Registry.
Part 3
Results
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