Team:Baltimore US/Project

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The first regional group within the DIY-Bio Community to secure entrance into the iGEM competition. In the years to come we hope other DIY-Bio communities will form alliances with local institutions of learning so they also may be able to help us form a sub-category or alternative competition/collaborations within the National iGEM Jamborees.
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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 tradgedy.

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 feedforward net with back-propogation 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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Contents

Overall project

Our Project consists of 3 categories of intended focus.

1) DIY-Gem Educational Focus. Our team being made up of a number of professionals with varying backgrounds and mixed depth of biological understanding wish to make a series of educational tools that will provide enhanced understanding and accessibility to the beginner experimenting with Synthetic Biology techniques.

2) DIY-Gem Tools. Creation of Bb Parts that will self-replicate several of the more expensive enzymes necessary throughout the processes utilized in Synthetic Biology with particular interest in the Bb Specific tools. Analysis of effectiveness and purifications for the do it yourself minded lab. Creation of various essential hardware components, with open-source dissemination of plans.

3) Artificial Neural Network. Creation of key tools required to advance the depth of bacterial problem solvers. Long term project to build upon some earlier research with key-lock systems created by Berkeley.

== 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.MolecularCloning.com

We then used the Gene Designer 2.0 freeware from DNA2.0 (https://www.dna20.com/tools/genedesigner.php) - 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 - http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

PolI Coli Primers For Overlap Extension PCR

PCR Reaction - 1

Fwd PolI Complement (56.5% CG Complement / 63.6 c)
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC
60.5 c ; 56.5 % GC Concetration

TAQ Rm
CTCCCGGTACTGAAGGATCTTCTCCAC
61.5 c ; 55.6 % GC Concentration

PCR 2 Reaction

TAQ Fm
GTGGAGAAGATCCTTCAGTACCGGGAG
61.5 c; 55.6 % GC

Bb Suffix + TAQ (Reverse Complement)
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC
61.8 c; 65 % GC

PCR 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 with

<partinfo>R0010 - Promoter (LacI)</partinfo>
<partinfo>B0034 - Strong RBS</partinfo>
NEW PART - PolI Bb Format</partinfo>
<partinfo>B0015 - Double Terminator
Psb1_?_3 - Plasmid of Interest with Chosen Resistance


<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

---
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)

Whallah!!! Brand New Taq Polymerase Bb Part.

Part 2

The Experiments

Part 3

Results

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