http://2010.igem.org/wiki/index.php?title=Special:Contributions/MPekala&feed=atom&limit=50&target=MPekala&year=&month=2010.igem.org - User contributions [en]2024-03-29T09:10:02ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Baltimore_US/Notebook/DIYEquipmentTeam:Baltimore US/Notebook/DIYEquipment2010-10-28T03:16:35Z<p>MPekala: /* Designs */</p>
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__NOTOC__<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<BR><br />
== DIY Equipment Notes ==<br />
==== Designs ====<br />
<br />
Semi-Final Equipment Mockups<br> <br />
[[image:DIYelectrophoresis1.JPG|300px]] [[image:DIYelectrophoresis2.JPG|300px]]<br />
<br />
First design integrating power and control onto the same board. Worked but we decided to seperate the power and the control for safety and cost reasons.<br />
[[image:Baltimore_US_EPDesign1.jpg|300px]]<br />
<br />
Several designs we made separated power and control. The first three of these were laid out several times for ease of construction. The designs worked, however only for the electrophoresis. They consisted of a simple bridge circuit, voltage sensor, and a micro controller driving the circuit. A design decision was made to attempt to get the electrophoresis and the PCR working with the same control electronics. This meant that since we were going to use a Peltier device for the PCR, we could not use a simple bridge like we used in this design. <br><br />
[[image:Baltimore_US_EPDesign2.jpg|300px]][[image:Baltimore_US_EPBoard2.jpg|300px]]<br><br />
<br />
For the final design we decided to separate the main controller from the bridges completely. We added a low power H-Bridge to heat the lid of the PCR device and added a temperature sensor to allow us to read the PCR temperature and implement a simple control loop.<br />
<br><br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/DIYEquipmentTeam:Baltimore US/Notebook/DIYEquipment2010-10-28T03:13:55Z<p>MPekala: /* Designs */</p>
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!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
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|}<br />
__NOTOC__<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<BR><br />
== DIY Equipment Notes ==<br />
==== Designs ====<br />
<br />
Semi-Final Equipment Mockups<br> <br />
[[image:DIYelectrophoresis1.JPG|300px]] [[image:DIYelectrophoresis2.JPG|300px]]<br />
<br />
First design integrating power and control onto the same board. Worked but we decided to seperate the power and the control for safety and cost reasons.<br />
[[image:Baltimore_US_EPDesign1.jpg|300px]]<br />
<br />
Several designs we made separated power and control. The first three of these were laid out several times for ease of construction. The designs worked, however only for the electrophoresis. They consisted of a simple bridge circuit, voltage sensor, and a micro controller driving the circuit. A design decision was made to attempt to get the electrophoresis and the PCR working with the same control electronics. This meant that since we were going to use a Peltier device for the PCR, we could not use a simple bridge like we used in this design. <br><br />
[[image:Baltimore_US_EPDesign2.jpg|300px]][[image:Baltimore_US_EPBoard2.jpg|300px]]<br><br />
<br />
For the final design we decided to separate the main controller from the bridges completely. We added a low power H-Bridge to heat the lid of the PCR device and added a temperature sensor to allow us to read the PCR temperature and implement a simple control loop.<br />
[[image:Baltimore_US_F1.jpg|300px]][[image:Baltimore_US_F3.jpg|300px]][[image:Baltimore_US_F3.jpg|300px]]<br><br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/DIYEquipmentTeam:Baltimore US/Notebook/DIYEquipment2010-10-28T03:12:27Z<p>MPekala: /* Designs */</p>
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|}<br />
__NOTOC__<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<BR><br />
== DIY Equipment Notes ==<br />
==== Designs ====<br />
<br />
Semi-Final Equipment Mockups<br> <br />
[[image:DIYelectrophoresis1.JPG|300px]] [[image:DIYelectrophoresis2.JPG|300px]]<br />
<br />
First design integrating power and control onto the same board. Worked but we decided to seperate the power and the control for safety and cost reasons.<br />
[[image:Baltimore_US_EPDesign1.jpg|300px]]<br />
<br />
Several designs we made separated power and control. The first three of these were laid out several times for ease of construction. The designs worked, however only for the electrophoresis. They consisted of a simple bridge circuit, voltage sensor, and a micro controller driving the circuit. A design decision was made to attempt to get the electrophoresis and the PCR working with the same control electronics. This meant that since we were going to use a Peltier device for the PCR, we could not use a simple bridge like we used in this design. <br><br />
[[image:Baltimore_US_EPDesign2.jpg|300px]][[image:Baltimore_US_EPBoard2.jpg|300px]]<br><br />
<br />
For the final design we decided to separate the main controller from the bridges completely. We added a low power H-Bridge to heat the lid of the PCR device and added a temperature sensor to allow us to read the PCR temperature and implement a simple control loop.<br />
<br />
<br />
<br />
|}</div>MPekalahttp://2010.igem.org/File:Baltimore_US_EPBoard2.jpgFile:Baltimore US EPBoard2.jpg2010-10-28T03:10:56Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/DIYEquipmentTeam:Baltimore US/Notebook/DIYEquipment2010-10-28T03:10:29Z<p>MPekala: /* Designs */</p>
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!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
__NOTOC__<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<BR><br />
== DIY Equipment Notes ==<br />
==== Designs ====<br />
<br />
Semi-Final Equipment Mockups<br> <br />
[[image:DIYelectrophoresis1.JPG|300px]] [[image:DIYelectrophoresis2.JPG|300px]]<br />
<br />
First design integrating power and control onto the same board. Worked but we decided to seperate the power and the control for safety and cost reasons.<br />
[[image:Baltimore_US_EPDesign1.jpg|300px]]<br />
<br />
Several designs we made separated power and control. The first three of these were laid out several times for ease of construction. The designs worked, however only for the electrophoresis. They consisted of a simple bridge circuit, voltage sensor, and a micro controller driving the circuit. A design decision was made to attempt to get the electrophoresis and the PCR working with the same control electronics. This meant that since we were going to use a Peltier device for the PCR, we could not use a simple bridge like we used in this design. <br><br />
[[image:Baltimore_US_EPDesign2.jpg|300px]]<br><br />
[[image:Baltimore_US_EPBoard2.jpg|300px]]<br />
<br />
For the final design we decided to separate the main controller from the bridges completely. We added a low power H-Bridge to heat the lid of the PCR device and added a temperature sensor to allow us to read the PCR temperature and implement a simple control loop.<br />
<br />
<br />
<br />
|}</div>MPekalahttp://2010.igem.org/File:Baltimore_US_EPDesign2.jpgFile:Baltimore US EPDesign2.jpg2010-10-28T03:09:21Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Baltimore_US_EPDesign1.jpgFile:Baltimore US EPDesign1.jpg2010-10-28T03:07:39Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_US/ProjectTeam:Baltimore US/Project2010-10-28T00:27:42Z<p>MPekala: /* Developing low-cost alternatives to existing hardware: Project Details and Results */</p>
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__NOTOC__<br />
|<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== DIY-GEM: a path towards low cost high throughput gene synthesis. ==<br />
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."<br />
<br />
==Developing low-cost alternatives to existing enzymes: ''Taq'' polymerase Project Details==<br />
<br />
Thermus Aquaticus Polymerase I<br><br />
PolI<br><br />
J04639.1<br><br />
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128<br><br />
<br><br />
1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC<br><br />
51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG<br><br />
101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC<br><br />
151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT<br><br />
201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG<br><br />
251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC<br><br />
301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC<br><br />
351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC<br><br />
401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC<br><br />
451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA<br><br />
501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG<br><br />
551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG<br><br />
601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA<br><br />
651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC<br><br />
701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG<br><br />
751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC<br><br />
801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG<br><br />
851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA<br><br />
901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA<br><br />
951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC<br><br />
1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG<br><br />
1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC<br><br />
1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG<br><br />
1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC<br><br />
1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC<br><br />
1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT<br><br />
1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG<br><br />
1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT<br><br />
1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA<br><br />
1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC<br><br />
1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT<br><br />
1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC<br><br />
1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC<br><br />
1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC<br><br />
1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA<br><br />
1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC<br><br />
1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC<br><br />
1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA<br><br />
1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC<br><br />
1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA<br><br />
2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA<br><br />
2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG<br><br />
2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG<br><br />
2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG<br><br />
2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC<br><br />
2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG<br><br />
2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG<br><br />
2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC<br><br />
2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC<br><br />
2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG<br><br />
2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG<br><br />
2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG<br><br />
2601 GCTCTCCGCC AAGGAGTGAT ACCACC<br><br />
<br><br />
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/<br />
<br><br />
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. <br><br />
<br />
====Problem: PstI restriction site - Found @ 1717====<br />
CTGCAG-PstI restriction site<br><br />
GACGTC-Complement<br><br />
Solution - Site-specific Mutagenesis by Overlap Extension (see 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)<br />
<br><br><br />
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.<br><br />
<br />
====Primer Design====<br />
We designed two 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. <br><br />
GTGGAGAAGATCCT(T)CAGTACCGGCGG<br><br />
CACCTCTTCTAGGA(A)GTCATGGCCGCC<br><br />
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 for designing the oligos needed to make a part. (http://partsregistry.org/Help:Promoters/Construction) <br><br />
<br><br />
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/)<br><br />
<br><br />
====PolI Coli Primers For Overlap Extension PCR====<br />
'''PCR Reaction 1''' <br><br />
<br><br />
Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)<br><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC<br><br />
60.5 c ; 56.5 % GC Concetration<br><br />
<br><br />
TAQ Rm<br><br />
CTCCCGGTACTGAAGGATCTTCTCCAC<br><br />
61.5 c ; 55.6 % GC Concentration<br><br />
<br><br />
'''PCR Reaction - 2'''<br><br />
<br><br />
TAQ Fm<br><br />
GTGGAGAAGATCCTTCAGTACCGGGAG<br><br />
61.5 c; 55.6 % GC<br><br />
<br><br />
Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.<br><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC<br><br />
61.8 c; 65 % GC<br><br />
<br><br />
'''PCR Reaction - 3'''<br><br />
Bb Prefix & Suffix Primers<br><br />
<br><br />
Resuspend in 100 uL of H2O<br><br />
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)<br><br />
<br><br />
'''NEXT'''<br><br />
- Create Full Bb Prmr w Plasmid combining new part using<br><br />
<br><br />
<partinfo>R0010</partinfo> - Promoter (LacI)<br><br />
<partinfo>B0034</partinfo> - Strong RBS<br><br />
NEW PART - PolI Bb Format<br><br />
<partinfo>B0015</partinfo> - Double Terminator<br><br />
Psb1_?_3 - Plasmid of Interest with Chosen Resistance : http://partsregistry.org/Plasmid_backbones<br><br />
<br><br />
----<br />
<br><br />
<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS<br><br />
<br><br />
Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI<br><br />
Cut <partinfo>B0034</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because <br><br />
<br><br />
---<br><br />
New Part + <partinfo>B0015</partinfo> = New Part<br> <br />
<br><br />
Cut New Part w/EcoRI & SpeI<br><br />
Cut <partinfo>B0015</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
----<br><br />
<br><br />
Cut 1st Combined Part w/EcoRI & SpeI<br><br />
Cut 2nd Combined Part w/XbeI & PstI<br><br />
<br><br />
Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
'''Voila!!!''' Brand New Taq Polymerase Bb Part.<br><br />
<br />
<br />
<br />
== Developing low-cost alternatives to existing hardware: Project Details and Results ==<br />
An unfortunate fact of reality is that precision lab equipment is very costly. Even simple devices such as an Electrophoresis or PCR have significant cost. To ameliorate this a portion of our project will involve designing biological tools that are easy to build and are economical.<br><br><br />
<br />
[[Image:Baltimore US System.JPG|300px]]<br><br />
Our design incorporates two devices, a PCR and an Electrophoresis. Both are controlled by the same control electronics and power supply. A basic overview of the design can be seen in the diagram above. This design allows precise control from a computer or manual control from the control panel on the control electronics. Additionally multiple Electrophoresis devices can be controlled simultaneously in parallel and any power supply suitable can be used to power the devices. <br />
<br />
[[Image:EP.jpg|300px]]<br><br />
With regards to equipment, we have successfully constructed a very low-cost Gel Electrophoresis device and are currently working on the control software and control electronics. Additionally, we are working on getting a low-cost PCR thermocycler up and running as well.<br> [[Team:Baltimore_US/Notebook/EPInstructions|Instructions and Design files for building an Electrophoresis device]]</div>MPekalahttp://2010.igem.org/File:Baltimore_US_System.JPGFile:Baltimore US System.JPG2010-10-28T00:20:08Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/DIYEquipmentTeam:Baltimore US/Notebook/DIYEquipment2010-10-27T22:22:28Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
__NOTOC__<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<BR><br />
== DIY Equipment Notes ==<br />
==== Designs ====<br />
<br />
Semi-Final Equipment Mockups<br> <br />
[[image:DIYelectrophoresis1.JPG|300px]] [[image:DIYelectrophoresis2.JPG|300px]]<br />
<br />
First design integrating power and control onto the same board. Worked but we decided to seperate the power and the control for safety and cost reasons.<br />
[[image:Baltimore_US_EPDesign1.jpg|300px]]<br />
<br />
Several designs we made separated power and control. The first three of these were laid out several times for ease of construction. The designs worked, however only for the electrophoresis. They consisted of a simple bridge circuit, voltage sensor, and a micro controller driving the circuit. A design decision was made to attempt to get the electrophoresis and the PCR working with the same control electronics. This meant that since we were going to use a Peltier device for the PCR, we could not use a simple bridge like we used in this design.<br />
[[image:Baltimore_US_EPDesign2.jpg|300px]]<br />
<br />
For the final design we decided to separate the main controller from the bridges completely. We added a low power H-Bridge to heat the lid of the PCR device and added a temperature sensor to allow us to read the PCR temperature and implement a simple control loop.<br />
<br />
<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T21:41:30Z<p>MPekala: /* Materials and Tools */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<br />
== Developing low-cost alternatives to existing hardware: Electrophoresis Apparatus==<br />
To construct the cheap electrophoresis that we have designed you need several parts. All of the components of the electrophoresis are laser cut acrylic of .220" thickness. The design can be modified to use any thickness that is around .220". If you do not possess your own laser cutter or do not have access to one, the parts can be laser cut using several online services such as Ponoko (http://www.ponoko.com/), Pololu (http://www.pololu.com/catalog/product/749), or any other laser cutting service. Note that this does increase the price of the electrophoresis as can be expected from any small quantity machine shop. The design files and list of materials are included at the bottom of this page.<br />
<br />
==== Parts ====<br />
The following parts are needed:<br />
<gallery><br />
Image:Baltimore US Parts1.jpg |Holder Sides <br />
Image:Baltimore US Parts2.jpg |Anode/Cathode Holders<br />
Image:Baltimore US Parts3.jpg |Lid with Interlock<br />
Image:Baltimore US Parts4.jpg |Base <br />
Image:Baltimore US Parts5.jpg |Long Sides<br />
Image:Glue1.jpg |Plastruct weld<br />
Image:Glue2.jpg |Silicone Caulk <br />
Image:Baltimore_US_Not_Pictured.jpg |Conducting Plates<br />
</gallery><br />
<br />
<br />
==== Construction ====<br />
1. Get the two Holder sides and the base. <br><br />
[[Image:Build1.jpg|200px]]<br><br />
2. Glue the two Holder sides to the base using Plastruct Weld. Hold this together for five minutes. <br><br />
[[Image:Build2.jpg|200px]]<br><br />
3. If needed, brush more Plastruct weld into the cracks to ensure a seal. Hold this together. <br><br />
[[Image:Build3.jpg|200px]]<br><br />
4. Get the long sides and the previously constructed holder. <br><br />
[[Image:Build4.jpg|200px]]<br><br />
5. Glue the long sides to the previously constructed holder using Plastruct Weld. If needed brush more in the cracks.<br><br />
[[Image:Build5.jpg|200px]]<br><br />
6. Wait 24 hours for the Plastruct to set. <br><br />
7. Get the silicone caulk and the construct. <br><br />
[[Image:Build6.jpg|200px]]<br><br />
8. Caulk the construct on all of the inside seams. <br><br />
[[Image:Build7.jpg|200px]]<br><br />
9. Get the anode/cathode holders and conducting plates. Screw a plate onto the anode and cathode. <br><br />
[[Image:Build8.jpg|200px]]<br><br />
<br />
<br />
==== Finished Device ====<br />
[[Image:EP.jpg|400px]] [[Image:EP2.jpg|400px]]<br />
<br />
<br />
==== Design Files ====<br />
<br><br />
[[Media:Baltimore US EPDesign.zip| Design files for Electrophoresis]] <br><br />
<br><br />
==== Materials and Tools ====<br />
'''Materials needed:'''<br />
# 1 Sheet of Acrylic .220" at least 12"x12" (http://www.mcmaster.com/#acrylic/=9gmzxa)<br />
# 1 vial of Plastruct Weld (http://www3.towerhobbies.com/cgi-bin/wti0001p?&I=LXDJ94&P=ML)<br />
# 1 tube of Silicone Caulk (http://www.homedepot.com/h_d1/N-5yc1vZ1xiy/R-100004845/h_d2/ProductDisplay?langId=-1&storeId=10051&catalogId=10053)<br />
# 2 Bananna Plug Connectors {http://www.mouser.com/ProductDetail/Pomona-Electronics/1581-2/?qs=sGAEpiMZZMtAYTMy7wxAryRi8wdtadCi2Nvox40dylM%3d)<br />
# 2 3"x 2.5" x .015" stainless steel sheet (http://www.onlinemetals.com/merchant.cfm?pid=9857&step=4&showunits=inches&id=325&top_cat=1)<br />
Note that some of these materials will provide a great deal more then is needed for the construction of one device (The plastruct weld, Silicone Caulk, and Stainless Sheet are enough for approximately 15 devices). Additionally, all of the parts except the Bandanna Plug connectors can be replaced with generic equivalents. <br />
<br />
'''Tools Needed:'''<br />
# Laser Cutter<br />
# Tin Snips or Break/Shear<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T21:38:45Z<p>MPekala: /* Design Files */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<br />
== Developing low-cost alternatives to existing hardware: Electrophoresis Apparatus==<br />
To construct the cheap electrophoresis that we have designed you need several parts. All of the components of the electrophoresis are laser cut acrylic of .220" thickness. The design can be modified to use any thickness that is around .220". If you do not possess your own laser cutter or do not have access to one, the parts can be laser cut using several online services such as Ponoko (http://www.ponoko.com/), Pololu (http://www.pololu.com/catalog/product/749), or any other laser cutting service. Note that this does increase the price of the electrophoresis as can be expected from any small quantity machine shop. The design files and list of materials are included at the bottom of this page.<br />
<br />
==== Parts ====<br />
The following parts are needed:<br />
<gallery><br />
Image:Baltimore US Parts1.jpg |Holder Sides <br />
Image:Baltimore US Parts2.jpg |Anode/Cathode Holders<br />
Image:Baltimore US Parts3.jpg |Lid with Interlock<br />
Image:Baltimore US Parts4.jpg |Base <br />
Image:Baltimore US Parts5.jpg |Long Sides<br />
Image:Glue1.jpg |Plastruct weld<br />
Image:Glue2.jpg |Silicone Caulk <br />
Image:Baltimore_US_Not_Pictured.jpg |Conducting Plates<br />
</gallery><br />
<br />
<br />
==== Construction ====<br />
1. Get the two Holder sides and the base. <br><br />
[[Image:Build1.jpg|200px]]<br><br />
2. Glue the two Holder sides to the base using Plastruct Weld. Hold this together for five minutes. <br><br />
[[Image:Build2.jpg|200px]]<br><br />
3. If needed, brush more Plastruct weld into the cracks to ensure a seal. Hold this together. <br><br />
[[Image:Build3.jpg|200px]]<br><br />
4. Get the long sides and the previously constructed holder. <br><br />
[[Image:Build4.jpg|200px]]<br><br />
5. Glue the long sides to the previously constructed holder using Plastruct Weld. If needed brush more in the cracks.<br><br />
[[Image:Build5.jpg|200px]]<br><br />
6. Wait 24 hours for the Plastruct to set. <br><br />
7. Get the silicone caulk and the construct. <br><br />
[[Image:Build6.jpg|200px]]<br><br />
8. Caulk the construct on all of the inside seams. <br><br />
[[Image:Build7.jpg|200px]]<br><br />
9. Get the anode/cathode holders and conducting plates. Screw a plate onto the anode and cathode. <br><br />
[[Image:Build8.jpg|200px]]<br><br />
<br />
<br />
==== Finished Device ====<br />
[[Image:EP.jpg|400px]] [[Image:EP2.jpg|400px]]<br />
<br />
<br />
==== Design Files ====<br />
<br><br />
[[Media:Baltimore US EPDesign.zip| Design files for Electrophoresis]] <br><br />
<br><br />
==== Materials and Tools ====<br />
'''Materials needed:'''<br />
# 1 Sheet of Acrylic .220" at least 12"x12" (http://www.mcmaster.com/#acrylic/=9gmzxa)<br />
# 1 vial of Plastruct Weld (http://www3.towerhobbies.com/cgi-bin/wti0001p?&I=LXDJ94&P=ML)<br />
# 1 tube of Silicone Caulk (http://www.homedepot.com/h_d1/N-5yc1vZ1xiy/R-100004845/h_d2/ProductDisplay?langId=-1&storeId=10051&catalogId=10053)<br />
# 2 Bananna Plug Connectors {http://www.mouser.com/ProductDetail/Pomona-Electronics/1581-2/?qs=sGAEpiMZZMtAYTMy7wxAryRi8wdtadCi2Nvox40dylM%3d)<br />
# 2 3"x 2.5" x .015" stainless steel sheet (http://www.onlinemetals.com/merchant.cfm?pid=9857&step=4&showunits=inches&id=325&top_cat=1)<br />
Note that some of these materials will provide a great deal more then is needed for the construction of one device. Additionally, all of the parts except the Bandanna Plug connectors can be replaced with generic equivalents. <br />
<br />
'''Tools Needed:'''<br />
# Laser Cutter<br />
# Tin Snips<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T18:27:13Z<p>MPekala: /* Design Files */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<br />
== Building a cheap Electrophoresis ==<br />
To construct the cheap electrophoresis that we have designed you need several parts. All of the components of the electrophoresis are laser cut acrylic of .220" thickness. The design can be modified to use any thickness that is around .220". If you do not possess your own laser cutter or do not have access to one, the parts can be laser cut using several online services such as Ponoko (http://www.ponoko.com/), Pololu (http://www.pololu.com/catalog/product/749), or any other laser cutting service. Note that this does increase the price of the electrophoresis as can be expected from any small quantity machine shop. The design files and list of materials are included at the bottom of this page.<br />
<br />
==== Parts ====<br />
The following parts are needed:<br />
<gallery><br />
Image:Baltimore US Parts1.jpg |Holder Sides <br />
Image:Baltimore US Parts2.jpg |Anode/Cathode Holders<br />
Image:Baltimore US Parts3.jpg |Lid with Interlock<br />
Image:Baltimore US Parts4.jpg |Base <br />
Image:Baltimore US Parts5.jpg |Long Sides<br />
Image:Glue1.jpg |Plastruct weld<br />
Image:Glue2.jpg |Silicone Caulk <br />
Image:Baltimore_US_Not_Pictured.jpg |Conducting Plates<br />
</gallery><br />
<br />
<br />
==== Construction ====<br />
1. Get the two Holder sides and the base. <br><br />
[[Image:Build1.jpg|200px]]<br><br />
2. Glue the two Holder sides to the base using Plastruct Weld. Hold this together for five minutes. <br><br />
[[Image:Build2.jpg|200px]]<br><br />
3. If needed, brush more Plastruct weld into the cracks to ensure a seal. Hold this together. <br><br />
[[Image:Build3.jpg|200px]]<br><br />
4. Get the long sides and the previously constructed holder. <br><br />
[[Image:Build4.jpg|200px]]<br><br />
5. Glue the long sides to the previously constructed holder using Plastruct Weld. If needed brush more in the cracks.<br><br />
[[Image:Build5.jpg|200px]]<br><br />
6. Wait 24 hours for the Plastruct to set. <br><br />
7. Get the silicone caulk and the construct. <br><br />
[[Image:Build6.jpg|200px]]<br><br />
8. Caulk the construct on all of the inside seams. <br><br />
[[Image:Build7.jpg|200px]]<br><br />
9. Get the anode/cathode holders and conducting plates. Screw a plate onto the anode and cathode. <br><br />
[[Image:Build8.jpg|200px]]<br><br />
<br />
<br />
==== Finished Device ====<br />
[[Image:EP.jpg|400px]] [[Image:EP2.jpg|400px]]<br />
<br />
<br />
==== Design Files ====<br />
[[Media:Baltimore US EPDesign.zip| Design files for Electrophoresis]]<br />
|}</div>MPekalahttp://2010.igem.org/File:Baltimore_US_EPDesign.zipFile:Baltimore US EPDesign.zip2010-10-27T18:23:29Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T17:35:24Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DYI-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' Polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' 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. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue where the community could step into this competition, gain hands-on experience, and learn the tools, techniques, and resources necessary to explore this emerging field under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" align="center"<br />
|<br />
<center>'''Sponsors:'''</center><br />
[[image:Qiagen logo.png|150px]] [[image:Axygen logo.png|150px]][[image:NEB logo.jpg|150px]]<br />
<br />
|}</div>MPekalahttp://2010.igem.org/File:NEB_logo.jpgFile:NEB logo.jpg2010-10-27T17:33:03Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Axygen_logo.pngFile:Axygen logo.png2010-10-27T17:32:10Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T17:28:52Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DYI-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' Polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' 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. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue where the community could step into this competition, gain hands-on experience, and learn the tools, techniques, and resources necessary to explore this emerging field under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T16:43:15Z<p>MPekala: /* Building a cheap Electrophoresis */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<br />
== Building a cheap Electrophoresis ==<br />
To construct the cheap electrophoresis that we have designed you need several parts. All of the components of the electrophoresis are laser cut acrylic of .220" thickness. The design can be modified to use any thickness that is around .220". If you do not possess your own laser cutter or do not have access to one, the parts can be laser cut using several online services such as Ponoko (http://www.ponoko.com/), Pololu (http://www.pololu.com/catalog/product/749), or any other laser cutting service. Note that this does increase the price of the electrophoresis as can be expected from any small quantity machine shop. The design files and list of materials are included at the bottom of this page.<br />
<br />
==== Parts ====<br />
The following parts are needed:<br />
<gallery><br />
Image:Baltimore US Parts1.jpg |Holder Sides <br />
Image:Baltimore US Parts2.jpg |Anode/Cathode Holders<br />
Image:Baltimore US Parts3.jpg |Lid with Interlock<br />
Image:Baltimore US Parts4.jpg |Base <br />
Image:Baltimore US Parts5.jpg |Long Sides<br />
Image:Glue1.jpg |Plastruct weld<br />
Image:Glue2.jpg |Silicone Caulk <br />
Image:Baltimore_US_Not_Pictured.jpg |Conducting Plates<br />
</gallery><br />
<br />
<br />
==== Construction ====<br />
1. Get the two Holder sides and the base. <br><br />
[[Image:Build1.jpg|200px]]<br><br />
2. Glue the two Holder sides to the base using Plastruct Weld. Hold this together for five minutes. <br><br />
[[Image:Build2.jpg|200px]]<br><br />
3. If needed, brush more Plastruct weld into the cracks to ensure a seal. Hold this together. <br><br />
[[Image:Build3.jpg|200px]]<br><br />
4. Get the long sides and the previously constructed holder. <br><br />
[[Image:Build4.jpg|200px]]<br><br />
5. Glue the long sides to the previously constructed holder using Plastruct Weld. If needed brush more in the cracks.<br><br />
[[Image:Build5.jpg|200px]]<br><br />
6. Wait 24 hours for the Plastruct to set. <br><br />
7. Get the silicone caulk and the construct. <br><br />
[[Image:Build6.jpg|200px]]<br><br />
8. Caulk the construct on all of the inside seams. <br><br />
[[Image:Build7.jpg|200px]]<br><br />
9. Get the anode/cathode holders and conducting plates. Screw a plate onto the anode and cathode. <br><br />
[[Image:Build8.jpg|200px]]<br><br />
<br />
<br />
==== Finished Device ====<br />
[[Image:EP.jpg|400px]] [[Image:EP2.jpg|400px]]<br />
<br />
<br />
==== Design Files ====<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T16:42:58Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
== Building a cheap Electrophoresis ==<br />
To construct the cheap electrophoresis that we have designed you need several parts. All of the components of the electrophoresis are laser cut acrylic of .220" thickness. The design can be modified to use any thickness that is around .220". If you do not possess your own laser cutter or do not have access to one, the parts can be laser cut using several online services such as Ponoko (http://www.ponoko.com/), Pololu (http://www.pololu.com/catalog/product/749), or any other laser cutting service. Note that this does increase the price of the electrophoresis as can be expected from any small quantity machine shop. The design files and list of materials are included at the bottom of this page.<br />
<br />
==== Parts ====<br />
The following parts are needed:<br />
<gallery><br />
Image:Baltimore US Parts1.jpg |Holder Sides <br />
Image:Baltimore US Parts2.jpg |Anode/Cathode Holders<br />
Image:Baltimore US Parts3.jpg |Lid with Interlock<br />
Image:Baltimore US Parts4.jpg |Base <br />
Image:Baltimore US Parts5.jpg |Long Sides<br />
Image:Glue1.jpg |Plastruct weld<br />
Image:Glue2.jpg |Silicone Caulk <br />
Image:Baltimore_US_Not_Pictured.jpg |Conducting Plates<br />
</gallery><br />
<br />
<br />
==== Construction ====<br />
1. Get the two Holder sides and the base. <br><br />
[[Image:Build1.jpg|200px]]<br><br />
2. Glue the two Holder sides to the base using Plastruct Weld. Hold this together for five minutes. <br><br />
[[Image:Build2.jpg|200px]]<br><br />
3. If needed, brush more Plastruct weld into the cracks to ensure a seal. Hold this together. <br><br />
[[Image:Build3.jpg|200px]]<br><br />
4. Get the long sides and the previously constructed holder. <br><br />
[[Image:Build4.jpg|200px]]<br><br />
5. Glue the long sides to the previously constructed holder using Plastruct Weld. If needed brush more in the cracks.<br><br />
[[Image:Build5.jpg|200px]]<br><br />
6. Wait 24 hours for the Plastruct to set. <br><br />
7. Get the silicone caulk and the construct. <br><br />
[[Image:Build6.jpg|200px]]<br><br />
8. Caulk the construct on all of the inside seams. <br><br />
[[Image:Build7.jpg|200px]]<br><br />
9. Get the anode/cathode holders and conducting plates. Screw a plate onto the anode and cathode. <br><br />
[[Image:Build8.jpg|200px]]<br><br />
<br />
<br />
==== Finished Device ====<br />
[[Image:EP.jpg|400px]] [[Image:EP2.jpg|400px]]<br />
<br />
<br />
==== Design Files ====<br />
<br />
|}</div>MPekalahttp://2010.igem.org/File:Baltimore_US_Parts5.jpgFile:Baltimore US Parts5.jpg2010-10-27T16:12:40Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Baltimore_US_Parts4.jpgFile:Baltimore US Parts4.jpg2010-10-27T16:12:25Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Baltimore_US_Parts3.jpgFile:Baltimore US Parts3.jpg2010-10-27T16:12:05Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Baltimore_US_Parts2.jpgFile:Baltimore US Parts2.jpg2010-10-27T16:11:49Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Baltimore_US_Parts1.jpgFile:Baltimore US Parts1.jpg2010-10-27T16:07:32Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:PartsOverview.jpgFile:PartsOverview.jpg2010-10-27T16:06:21Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Glue2.jpgFile:Glue2.jpg2010-10-27T16:06:06Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Glue1.jpgFile:Glue1.jpg2010-10-27T16:05:05Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T16:04:48Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
[[Image:Build1.jpg]]<br />
<br />
[[Image:Build2.jpg]]<br />
<br />
[[Image:Build3.jpg]]<br />
<br />
[[Image:Build4.jpg]]<br />
<br />
[[Image:Build5.jpg]]<br />
<br />
[[Image:Build6.jpg]]<br />
<br />
[[Image:Build7.jpg]]<br />
<br />
[[Image:Build8.jpg]]<br />
<br />
<br />
[[Image:EP.jpg]]<br />
<br />
<br />
[[Image:EP2.jpg]]<br />
|}</div>MPekalahttp://2010.igem.org/File:EP.jpgFile:EP.jpg2010-10-27T16:03:26Z<p>MPekala: uploaded a new version of "Image:EP.jpg"</p>
<hr />
<div>Finished Electrophoresis Device</div>MPekalahttp://2010.igem.org/File:EP2.jpgFile:EP2.jpg2010-10-27T16:03:06Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build8.jpgFile:Build8.jpg2010-10-27T16:02:35Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build7.jpgFile:Build7.jpg2010-10-27T16:02:17Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build6.jpgFile:Build6.jpg2010-10-27T16:01:39Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build5.jpgFile:Build5.jpg2010-10-27T16:01:28Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build4.jpgFile:Build4.jpg2010-10-27T16:01:16Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build3.jpgFile:Build3.jpg2010-10-27T16:00:58Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build2.jpgFile:Build2.jpg2010-10-27T16:00:38Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build1.jpgFile:Build1.jpg2010-10-27T15:59:33Z<p>MPekala: uploaded a new version of "Image:Build1.jpg"</p>
<hr />
<div></div>MPekalahttp://2010.igem.org/File:Build1.jpgFile:Build1.jpg2010-10-27T15:39:05Z<p>MPekala: </p>
<hr />
<div></div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T15:33:31Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
== Constructing a Cheap Electrophoresis ==<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T15:29:00Z<p>MPekala: New page: center {| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center" !align="center"|[[T...</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/ProjectTeam:Baltimore US/Project2010-10-27T15:27:16Z<p>MPekala: /* Results */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== DIY-GEM: a path towards low cost high throughput gene synthesis. ==<br />
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."<br />
<br />
==PoliColi Project Details==<br />
<br />
Thermus Aquaticus Polymerase I<br><br />
PolI<br><br />
J04639.1<br><br />
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128<br><br />
<br><br />
1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC<br><br />
51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG<br><br />
101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC<br><br />
151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT<br><br />
201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG<br><br />
251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC<br><br />
301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC<br><br />
351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC<br><br />
401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC<br><br />
451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA<br><br />
501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG<br><br />
551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG<br><br />
601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA<br><br />
651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC<br><br />
701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG<br><br />
751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC<br><br />
801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG<br><br />
851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA<br><br />
901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA<br><br />
951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC<br><br />
1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG<br><br />
1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC<br><br />
1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG<br><br />
1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC<br><br />
1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC<br><br />
1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT<br><br />
1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG<br><br />
1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT<br><br />
1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA<br><br />
1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC<br><br />
1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT<br><br />
1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC<br><br />
1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC<br><br />
1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC<br><br />
1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA<br><br />
1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC<br><br />
1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC<br><br />
1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA<br><br />
1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC<br><br />
1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA<br><br />
2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA<br><br />
2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG<br><br />
2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG<br><br />
2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG<br><br />
2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC<br><br />
2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG<br><br />
2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG<br><br />
2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC<br><br />
2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC<br><br />
2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG<br><br />
2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG<br><br />
2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG<br><br />
2601 GCTCTCCGCC AAGGAGTGAT ACCACC<br><br />
<br><br />
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/<br />
<br><br />
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. <br><br />
<br />
====Problem: PstI restriction site - Found @ 1717====<br />
CTGCAG-PstI restriction site<br><br />
GACGTC-Complement<br><br />
Solution - Site-specific Mutagenesis by Overlap Extension (see 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)<br />
<br><br><br />
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.<br><br />
<br />
====Primer Design====<br />
We designed two 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. <br><br />
GTGGAGAAGATCCT(T)CAGTACCGGCGG<br><br />
CACCTCTTCTAGGA(A)GTCATGGCCGCC<br><br />
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 for designing the oligos needed to make a part. (http://partsregistry.org/Help:Promoters/Construction) <br><br />
<br><br />
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/)<br><br />
<br><br />
====PolI Coli Primers For Overlap Extension PCR====<br />
'''PCR Reaction 1''' <br><br />
<br><br />
Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)<br><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC<br><br />
60.5 c ; 56.5 % GC Concetration<br><br />
<br><br />
TAQ Rm<br><br />
CTCCCGGTACTGAAGGATCTTCTCCAC<br><br />
61.5 c ; 55.6 % GC Concentration<br><br />
<br><br />
'''PCR Reaction - 2'''<br><br />
<br><br />
TAQ Fm<br><br />
GTGGAGAAGATCCTTCAGTACCGGGAG<br><br />
61.5 c; 55.6 % GC<br><br />
<br><br />
Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.<br><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC<br><br />
61.8 c; 65 % GC<br><br />
<br><br />
'''PCR Reaction - 3'''<br><br />
Bb Prefix & Suffix Primers<br><br />
<br><br />
Resuspend in 100 uL of H2O<br><br />
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)<br><br />
<br><br />
'''NEXT'''<br><br />
- Create Full Bb Prmr w Plasmid combining new part using<br><br />
<br><br />
<partinfo>R0010</partinfo> - Promoter (LacI)<br><br />
<partinfo>B0034</partinfo> - Strong RBS<br><br />
NEW PART - PolI Bb Format<br><br />
<partinfo>B0015</partinfo> - Double Terminator<br><br />
Psb1_?_3 - Plasmid of Interest with Chosen Resistance : http://partsregistry.org/Plasmid_backbones<br><br />
<br><br />
----<br />
<br><br />
<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS<br><br />
<br><br />
Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI<br><br />
Cut <partinfo>B0034</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because <br><br />
<br><br />
---<br><br />
New Part + <partinfo>B0015</partinfo> = New Part<br> <br />
<br><br />
Cut New Part w/EcoRI & SpeI<br><br />
Cut <partinfo>B0015</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
----<br><br />
<br><br />
Cut 1st Combined Part w/EcoRI & SpeI<br><br />
Cut 2nd Combined Part w/XbeI & PstI<br><br />
<br><br />
Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
'''Voila!!!''' Brand New Taq Polymerase Bb Part.<br><br />
<br />
== Results ==<br />
<br />
[[Image:EP.jpg|300px]]<br><br />
With regards to equipment we have successfully constructed a very low cost Gel Electrophoresis device and are currently working on the control software and control electronics. Additionally we are working on getting the a low cost PCR up and running as well.<br> [[Team:Baltimore_US/Notebook/EPInstructions|Instructions and Design files for building an Electrophoresis device]]</div>MPekalahttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T15:25:41Z<p>MPekala: /* Poli-Coli: a path towards low-cost high-throughput gene synthesis */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
'''Sponsors''' <br><br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DYI-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' Polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' 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. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue where the community could step into this competition, gain hands-on experience, and learn the tools, techniques, and resources necessary to explore this emerging field under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/TeamTeam:Baltimore US/Team2010-10-27T15:19:38Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br />
{| align="center"<br />
|<br />
|[[Image:Baltimore_US_team.png|400px]]<br />
|}<br />
<!--- The Mission, Experiments ---><br />
<br><br />
== '''Who we are''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Lab Manager/Advisor''': Thomas Burkett<br />
*'''Advisor 1''': Lisa Scheifele<br />
*'''Advisor 2''': T Elizabeth Goode <br />
<br><br />
'''Undergrads:'''<br />
*'''Student 1''': Ryan Ogle, LMT <br />
*'''Student 2''': Patrick O'Neil<br />
*'''Student 3''': Robert Buck <br />
*'''Student 4''': Miles Pekala<br />
*'''Student 5''': Gary Cygiel<br />
*'''Student 6''': Steven Stowell<br />
*'''Student 7''': Duke Kessler<br />
*'''Student 8''': Bernadette Gallagher<br />
<br />
|<br />
<gallery><br />
Image:Ryan.JPG|''Go Team Venture!'' <br><br>'''Ryan Ogle, LMT''' <br />
Image:Patrickmugshot.jpg|<br><br>'''Patrick O'Neill''' <br />
Image:Baltimore_US_Team_member_3.png|(Robert's mugshot here) <br><br>'''Robert Buck''' <br />
Image:Baltimore_US_Team_member_4.png|(Miles' mugshot here) <br><br>'''Miles Pekala''' <br />
Image:Baltimore_US_Team_member_5.png|(Gary's mugshot here) <br><br>'''Gary Cygiel''' <br />
Image:Baltimore_US_Team_member_6.png|(Steve's mugshot here) <br><br>'''Steven Stowell''' <br />
Image:Baltimore_US_Team_member_7.png|(Duke's mugshot here) <br><br>'''Duke Kessler''' <br />
Image:Bernadette_.JPG|(Bernadette's mugshot here) <br>'''Bernadette Gallagher''' <br />
Image:Baltimore_US_Team_member_9.png|(Tom's mugshot here) <br><br>'''Tom Burkett - Lab Manager/Advisor & Contact Person''' <br />
Image:Baltimore_US_Team__member_10.png|(Lisa's mugshot here) <br><br>'''Lisa Scheifele - Advisor''' <br />
Image:Baltimore_US_Team_member_11.png|(Liz's mugshot here )<br><br> '''Liz Goode - Advisor''' <br />
<br />
</gallery><br />
|}<br />
<br />
== '''What we did''' ==<br />
<br />
[[Image:DIYPatrick1.JPG|300px]] [[Image:DIYMiles.JPG|300px]] [[Image:DIYRobert.JPG|300px]]<br />
<br />
== '''Where we're from''' ==</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/TeamTeam:Baltimore US/Team2010-10-27T15:18:07Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br />
{| align="center"<br />
|<br />
|[[Image:Baltimore_US_team.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== '''Who we are''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Lab Manager/Advisor''': Thomas Burkett<br />
*'''Advisor 1''': Lisa Scheifele<br />
*'''Advisor 2''': T Elizabeth Goode <br />
<br />
<br />
'''Undergrads:'''<br />
<br />
*'''Student 1''': Ryan Ogle, LMT <br />
*'''Student 2''': Patrick O'Neil<br />
*'''Student 3''': Robert Buck <br />
*'''Student 4''': Miles Pekala<br />
*'''Student 5''': Gary Cygiel<br />
*'''Student 6''': Steven Stowell<br />
*'''Student 7''': Duke Kessler<br />
*'''Student 8''': Bernadette Gallagher<br />
<br />
<br />
|<br />
<gallery><br />
Image:Ryan.JPG|''Go Team Venture!'' <br><br>'''Ryan Ogle, LMT''' <br />
Image:Patrickmugshot.jpg|<br><br>'''Patrick O'Neill''' <br />
Image:Baltimore_US_Team_member_3.png|(Robert's mugshot here) <br><br>'''Robert Buck''' <br />
Image:Baltimore_US_Team_member_4.png|(Miles' mugshot here) <br><br>'''Miles Pekala''' <br />
Image:Baltimore_US_Team_member_5.png|(Gary's mugshot here) <br><br>'''Gary Cygiel''' <br />
Image:Baltimore_US_Team_member_6.png|(Steve's mugshot here) <br><br>'''Steven Stowell''' <br />
Image:Baltimore_US_Team_member_7.png|(Duke's mugshot here) <br><br>'''Duke Kessler''' <br />
Image:Bernadette_.JPG|(Bernadette's mugshot here) <br>'''Bernadette Gallagher''' <br />
Image:Baltimore_US_Team_member_9.png|(Tom's mugshot here) <br><br>'''Tom Burkett - Lab Manager/Advisor & Contact Person''' <br />
Image:Baltimore_US_Team__member_10.png|(Lisa's mugshot here) <br><br>'''Lisa Scheifele - Advisor''' <br />
Image:Baltimore_US_Team_member_11.png|(Liz's mugshot here )<br><br> '''Liz Goode - Advisor''' <br />
<br />
</gallery><br />
|}<br />
<br />
== '''What we did''' ==<br />
<br />
<br />
[[Image:DIYPatrick1.JPG|300px]]<br />
<br />
<br />
[[Image:DIYMiles.JPG|300px]]<br />
<br />
<br />
[[Image:DIYRobert.JPG|300px]]<br />
<br />
== '''Where we're from''' ==</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/TeamTeam:Baltimore US/Team2010-10-27T15:17:38Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br />
{|<br />
|<br />
|[[Image:Baltimore_US_team.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== '''Who we are''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
<br />
<br />
<br />
'''Advisors:'''<br />
<br />
*''' Lab Manager/Advisor''': Thomas Burkett<br />
*'''Advisor 1''': Lisa Scheifele<br />
*'''Advisor 2''': T Elizabeth Goode <br />
<br />
<br />
'''Undergrads:'''<br />
<br />
*'''Student 1''': Ryan Ogle, LMT <br />
*'''Student 2''': Patrick O'Neil<br />
*'''Student 3''': Robert Buck <br />
*'''Student 4''': Miles Pekala<br />
*'''Student 5''': Gary Cygiel<br />
*'''Student 6''': Steven Stowell<br />
*'''Student 7''': Duke Kessler<br />
*'''Student 8''': Bernadette Gallagher<br />
<br />
<br />
|<br />
<gallery><br />
Image:Ryan.JPG|''Go Team Venture!'' <br><br>'''Ryan Ogle, LMT''' <br />
Image:Patrickmugshot.jpg|<br><br>'''Patrick O'Neill''' <br />
Image:Baltimore_US_Team_member_3.png|(Robert's mugshot here) <br><br>'''Robert Buck''' <br />
Image:Baltimore_US_Team_member_4.png|(Miles' mugshot here) <br><br>'''Miles Pekala''' <br />
Image:Baltimore_US_Team_member_5.png|(Gary's mugshot here) <br><br>'''Gary Cygiel''' <br />
Image:Baltimore_US_Team_member_6.png|(Steve's mugshot here) <br><br>'''Steven Stowell''' <br />
Image:Baltimore_US_Team_member_7.png|(Duke's mugshot here) <br><br>'''Duke Kessler''' <br />
Image:Bernadette_.JPG|(Bernadette's mugshot here) <br>'''Bernadette Gallagher''' <br />
Image:Baltimore_US_Team_member_9.png|(Tom's mugshot here) <br><br>'''Tom Burkett - Lab Manager/Advisor & Contact Person''' <br />
Image:Baltimore_US_Team__member_10.png|(Lisa's mugshot here) <br><br>'''Lisa Scheifele - Advisor''' <br />
Image:Baltimore_US_Team_member_11.png|(Liz's mugshot here )<br><br> '''Liz Goode - Advisor''' <br />
<br />
</gallery><br />
|}<br />
<br />
== '''What we did''' ==<br />
<br />
<br />
[[Image:DIYPatrick1.JPG|300px]]<br />
<br />
<br />
[[Image:DIYMiles.JPG|300px]]<br />
<br />
<br />
[[Image:DIYRobert.JPG|300px]]<br />
<br />
== '''Where we're from''' ==</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/conjugationTeam:Baltimore US/Notebook/conjugation2010-10-27T05:30:00Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
__NOTOC__<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<br />
Some papers and notes Dr. Scheifele gathered on bacterial conjugation:<br />
<br />
[https://docs.google.com/leaf?id=1za21GHTa3HnOEnPdN1TLNdnFRZO3fS1stUeNgSAhgz2-j6POiaZYd8A9sT35&sort=name&layout=list&num=50 entry exclusion]<br />
<br />
[https://docs.google.com/leaf?id=1VIn-w1dmgPPfm0XgCtJpbKjE6L8sqIDUZUfGZdTBkw9Spr95z3b4Bcn1RIbf&sort=name&layout=list&num=50 retrotransfer or gene capture]<br />
<br />
[https://docs.google.com/leaf?id=1kojqOXSFHShqlxOyXK7tZUxYLycJ2W_WE71U--V44CmgVUkT60MIUJtDp7g8&sort=name&layout=list&num=50 Analysis of the Sequence and Gene Products of the Transfer]<br />
<br />
[https://docs.google.com/leaf?id=1hcOKhLwpcWsf9W9rwGEZ6MDdw3ttcG5r2rE0NnNifAvHAYSZG5fS8CEA7aqr&sort=name&layout=list&num=50s conjugation notes]<br />
<br />
Email Patrick if you can't access any of these.<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/Notebook/computationTeam:Baltimore US/Notebook/computation2010-10-27T05:29:47Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
__NOTOC__<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
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<br />
This page under construction. Meanwhile, check out:<br />
<br />
<ul><br />
<li>[[wikipedia:DNA computing| Wikipedia's DNA computing article]] </li><br />
<li>[http://arstechnica.com/reviews/2q00/dna/dna-1.html Ars Technica primer]</li><br />
<li>[http://www.cs.virginia.edu/~robins/Computing_with_DNA.pdf An article by Adleman written for a popular audience]</li><br />
<li>[http://crypto.stanford.edu/~dabo/biocomp.html Collection of more technical papers]</li><br />
</ul><br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/JuneGroupNotebookTeam:Baltimore US/JuneGroupNotebook2010-10-27T05:29:27Z<p>MPekala: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
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{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
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<br><br />
== Group Notebook for June 2010 ==<br />
__TOC__<br />
=== June 29th, 2010 ===<br />
In attendance: Day - Duke, Tom, Patrick, Ryan, Robert <br><br />
Evening: Gary, Liz, Tom, Patrick, Duke, Ryan <br><br />
<br><br />
During the day-shift Robert continued to work on the PCR optimization, with the second set of Gels showing a continued contamination of one of the main components, pointing possibly to the template mixing into the dNTP's. Patrick helped Tom with some Thermophilic cultures, and Ryan completed the Plasmid Transformations of T3, K3, & C3 along with the Batch prep of the CAT8 plasmids.<br><br />
<br><br />
We caught tonight's attendees up to speed as to where the group stands on it's individual projects. Then together we discussed in detail how to break up the foundational work-flow and what steps need to be accomplished to make them happen.<br />
Patrick detailed some of the foundational work previously performed by the earlier iGEM teams on Artificial Neural Networks.<br><br />
DIY-Gem notes: Creation of Pol-1 Taq Polmerase in Bb format J04637.1<br><br />
1. Amplify Pol-1 gene from T. Aquaticus <br><br />
Convert to Bb format - Add Bb Prefix and Suffix <br><br />
Change internal Pst-1 site - Single BP Mutation <br><br />
2. Characterize Part <br><br />
Error Rate? <br><br />
Activity <br><br />
Etc... (Versus Lab Ordered Purity) <br><br />
3. Educational Supplementation <br><br />
Techniques - How to do a single point mutation <br><br />
Tools Discussion / Software & Hardware <br><br />
<br><br />
DNA Computing Notes: Turing Automatons w. Back Propagation <br><br />
1. What Species is it? <br><br />
Identifying parts available. <br><br />
Identify parts to be made. <br><br />
Assemble parts. <br><br />
Build Assemblies. <br><br />
Test & Characterize System. <br><br />
What is a Neural Network? The XOR switching system. <br><br />
- Addressable Conjugation. <br><br />
- RNA Hairpins. <br><br />
- HSL Senders / Receivers. <br><br />
- Others. <br><br />
- Attaching varied weights among various pathways. <br><br />
- Summing. <br><br />
- Thresholdiing. <br><br />
- Back Propagation Control through Lysing (Cellular Suicide model). <br><br />
<br><br />
<br />
=== June 28th, 2010 ===<br />
In attendance: Day - Duke, Robert, Patrick, Robert, Ryan, Tom <br><br />
Evening: Miles, Robert, Patrick, Tom, Ryan, Burnadette <br><br />
<br><br />
We welcomed a new comer Bernadette Gallagher. And spoke more in depth about project design and workflow. Tom helped get our new team mate oriented, while Miles, Patrick and Robert worked on flushing out conceptual foundations for their projects of interest.<br><br />
<br><br />
Ryan - Performed Restrictions (e/p)and Ligations on Pb1t3,c3,k3 while assisting the batch prep upon the CAT8 plasmids, performed first step lysis to instruction 14 putting DNA in alcohol. <br><br />
<br />
=== June 24th, 2010 ===<br />
In Attendance: Patrick, Robert, Tom, Duke, Ryan <br><br />
<br><br />
After a roughly a week's break from official activities the team met to discuss<br />
strategies and tactics. <br><br />
A few hands have been taking up the lab tech training from Tom and Duke throughout the lazy summer days, while doing so they revisited the basic procedural steps of ligation, gel-phoresis, transformation, as well as the tasks<br />
of mixing and pouring media, basic chemical preparation, as well as safety and hygeine in the lab. Tom has also been introducing us to the concepts behind workflow and how to divide and stage the various processes towards a greater project concept. <br><br />
<br><br />
Miles suggested that he will be working towards several ideas in hardware project construction from a PCR unit to a DNA synthesizer. We discussed in depth Patrick's idea for the creation of a cellular automaton. He broke down the<br />
previous tactics in DNA computing where everything is synthesized then re-sequenced for the answer, then posited the creation of an Artificial Neural Network type approach in utilizing various signaling proteins to reach consensus<br />
and establishing some way to create back propagation of error. <br><br />
<br><br />
We've decided to restrict lab nights to Monday and Tuesday from 7-10pm, with a floater strategy meeting on Thursday nights so we can stimulate thought with a change of environment. With that in mind we are back tonight and look forward to<br />
seeing those of you who can make it in to keep cranking on our technique.<br><br />
<br><br />
<br />
=== June 16th, 2010 ===<br />
In attendance: Lisa, Ryan, Tom, Colin (for a short time), Duke (for a moment) <br><br />
Missing: Steve <br><br />
<br />
Earlier in the day, Robert and Ryan performed a mini-prep of the pcr'd plasmid backbones 3A1(2) and 5A1(2).<br />
Robert also began working on finding optimized conditions for pcr. <br><br />
<br />
With the small turnout, Ryan began the digestion restriction with Colin's assistance. We combined the 2 strains of 3A1 and 5A1 together, then put together 3 - .8 eppendorf's with 50 ul of rxn ingrediates. <br><br />
<br><br />
(2 tubes of positive control of both along with 1 negative control that contained no enzymes and 40 ul of H2O) <br><br />
<br><br />
5 ul of DNA <br><br />
1 ul of Pst <br><br />
1 ul of EcoR1 <br><br />
5 ul of Buffer <br><br />
38 ul of H20 <br><br />
<br><br />
50 ul - Negative Controls Labelled with minus sign.<br><br />
<br><br />
<br />
After preparation, the cells were put into the pcr block for a 30 minute heat cycle. <br><br />
Tom will be heading to Arizona, tomorrow, so we called it a night so he could pack and go. <br><br />
It has been suggested that we run a "Low-Heat Agar Gel" so we can find and physically splice out the appropriate sized plasmid structures. <br><br />
<br><br />
<br />
=== June 15th, 2010 ===<br />
In attendance: Patrick, Ryan, Duke, Tom, Steve <br><br />
Missing: Colin, Liz, Scott, Andy <br><br />
<br><br />
Tom says results seemed to have worked for all of the transformations this time. Some better then others. <br><br />
<br><br />
Steve asks the question.. "Why did transformation work better this time?" <br><br />
Tom discusses differences in attempts. <br><br />
<br><br />
Plasmid contains part a, another contains part b, another part c is simply a linear piece of DNA representing a Tet resistant backbone with the EcoRi and PstI restriction sites. <br><br />
<br><br />
We cut Part A with EcoR1 and Xba1. <br><br />
We cut Part B with Spe1 and Pst1. <br><br />
We cut the Linear Backbone Part C wit EcoR1 and Pst1. <br><br />
<br><br />
Then we combined everything into one tube using 2 ul of each part into 1 tube, and added t4 DNA ligase, hoping to get a Part A joined to Part B, because the Spe1 combines with the Xba1. And the Part A would join Part C with the EcoR1 and the Part B with Part C at the Pst1 site. <br><br />
<br><br />
(See photo of board - ) <br><br />
<br><br />
Last week, we got nothing, so why? <br><br />
Where could things go wrong? <br><br />
They could go wrong at the restriction digestion reaction (less likely as process is fairly robust, and had been tested the day previously.)<br><br />
<br><br />
Ligation reaction may not have worked.. (again less likely, process is fairly robust). If the buffer isn't mixed well it may separate. <br><br />
How do we test? we could perform pcr to see if the ligation took place, using primers to amplify the ligated segment checking with gel electrophoreis to identify. <br><br />
<br />
Transformation reaction - (Except we ran positive controls successfully) <br><br />
Still possible to have mixed up the media, since positive used Amp instead.. <br><br />
Tom checked the media and found that wasn't the case. <br><br />
Amount of Plasmid may not have been accurate. (The only thing we didn't physically check ahead of time.) <br><br />
<br><br />
Pure operator/Pipetting action - such as the 30 second heating followed by<br />
kill cycle. <br><br />
<br><br />
-------------- <br><br />
<br><br />
Detection limits for Ethidium Bromide 10-15 ngs of a product. <br><br />
(3600 bp ) (650 gm/b.p molar weight) <br><br />
2,340,000 Grams per mole of 3600 bp molecule. <br><br />
50 nanograms <br><br />
(50 x 10-9) / 2,340,000 = 2.1 x 10-14 moles <br><br />
<br><br />
Transformation rxn is 1 x 10 (-8/-9), still fairly efficient.<br><br />
<br><br />
We did 2 things last night, we did the transformations into the stocks.. <br><br />
<br><br />
By using assembly strategies that don't use the same resistance as the parts, we can more efficiently screen out non-transformed pieces. <br><br />
<br />
Previously in Genetic Engineering all parts and pieces had variorus resistances where as bb tries to create a standard. <br><br />
<br />
Cells can be lost in replication due to metabolic costs, and new generations lacking the Costly programming tend to outgrow the special cells. <br><br />
<br />
When designing the assembly strategy try to use a plasmid backbone with a different resistance to allow the filtration/screening of parts that didn't transform correctly.<br><br />
<br><br />
-----<br><br />
Gel-Electrophoresis Prep Review for tonights run. <br><br />
<br />
Agarose 2 % for smaller parts will create a more viscous environment. <br><br />
0.8% for the larger parts will allow longer molecules to move better. <br><br />
<br />
50ml final volume <br><br />
<br />
Weigh out the agarose (.4 gms for the 0.8%) (1 gm for the 2%) <br><br />
<br />
Emphasize again the importance of NCBI databases... http://www.ncbi.nlm.nih.gov/ <br><br />
With links to BLAST, PubMed, and other official literature. Program called entree which links all the information together. <br><br />
<br><br />
When reviewing the tubes from last evening left in PCR there was difficulty in identifying the tubes that were done by each individual. We spent a little time using the process of elimination. Knowing Patrick, Steve, Miles, Ryans and Gary's without issue, we had to guesstimate which was Roberts, and which was David's. The reason we needed differentiation was to create a loading template, in one of the two different agarose preparations. <br><br />
<br><br />
<br />
=== June 14, 2010 ===<br />
In Attendance: Tom, Duke, Patrick, Robert, Steve, Gary, David, Ryan, Miles<br />
<br />
3 Separate transformations attempted: <partinfo>pSB1C3</partinfo> [1-3A] (ChlorAmphenacol resistant), <partinfo>pSB1K3</partinfo> [1-5A](Kanamicin resistant), <partinfo>pSB1T3</partinfo> [1-7A] (Tetracycline resistant)<br />
Control Group: PET-17B (still had separate resistance for Ampicillan)<br />
Tom will be culturing some adjacently resistant control groups for our next trial, thereby eliminating the extra variable in our testing.<br />
<br />
<!-- Gary Cygiel --> <br />
Procedural experience: <br><br />
Transformations using 1-3A<br />
50uL EXPERIMENTAL <br><br />
200uL EXPERIMENTAL <br> <br />
250uL NEGATIVE CONTROL <br><br />
Cells were plated and put in the 37C incubator. Plates labeled in <font color="red">RED</font>.<br />
<br />
9 Parts for PCR - 7 Distributed amongst us. Various participants were asked to look up their function and various bp lengths.<br />
<br />
Patrick - <partinfo>Bba_R0010</partinfo> - 200 bp <br><br />
Gary - <partinfo>Bba_R0063</partinfo> - 151 bp <br><br />
Robert - <partinfo>Bba_J04450</partinfo> - 1,069 bp <br><br />
Dave - <partinfo>Bba_R0062</partinfo> - 55 bp<br><br />
Steve - <partinfo>Bba_J23009</partinfo> - 97 bp<br><br />
Miles - <partinfo>Bba_I731014</partinfo> 1,938 bp<br><br />
Ryan - <partinfo>Bba_I13507</partinfo> - Intermediate Screening Plasmid - 861 bp <br><br />
<br />
PCR for part [http://partsregistry.org/wiki/index.php/Part:BBa_R0063 R0063]: <br><br />
Vial labeled with <font color="red">RED X </font> is the EXPERIMENTAL REACTION <br><br />
Vial labeled with <font color="red">RED Y </font> is the TEMPLATE CONTROL <br><br />
Vial labeled with <font color="red">RED Z </font> is the PRIMER CONTROL <br><br />
<br />
PCR Polymerase Chain Reactions<br><br />
<br />
Requirements <br><br />
1. Template <br><br />
2. 2 Primers (limiting) <br><br />
3. dNTP's (limiting) <br><br />
4. Polymerase (limiting by heatcycle) Using Taq which tends to transcribe more errors. <br><br />
5. Buffer (mg++) (limiting) <br><br />
<br />
Primers are 18-30 bp long. Have to be at least 15 bp long. Primers bind to template DNA, one primer for each (5'/3' strands).<br />
Review: What is a base..? Sugar with A, C, T or G is a nucleotide. BP are the matching set of 5' and 3' hydrogen bonded nucleotides.<br />
<br />
Polymerase links free dNTP's to the opened strand edges of basepairs. <br><br />
Denaturation 95 - 98 degrees c... DNA Melts <br><br />
Anealing 40 - 65 degrees c... Primer Binds <br><br />
Extension 72 degrees c... Strands Extend <br><br />
30 second cycles... <br><br />
<br />
As the DNA heats it denatures then as it cools the primers anneal and polymerization takes place creating twice as many strands. The strands are held together only by hydrogen bonds, making it very easy to melt and reform. The polymerase acts like a little machine stitching the free dNTP's into the template.<br />
<br />
BioBrick plasmids have bb prefix and suffix. Primer binding sites vf2, vr.<br />
Theoretically any area can become a primer site.<br />
<br />
With PCR we can identify and amplify particular strands/compositions. You have to know how much of the requirements to use.<br />
Gary asked if Tom could show us where to find the optimized volume information at, and he agreed to pull out the research to show how to optimize.<br />
<br />
With PCR you can also design primers and introduce mutations. If primers are too short they can bind to the wrong spot.<br />
Actual reactions may vary based on bp length and combination. Usually they are tweaked for the optimal over a series of trials.<br />
<br />
PCR Reactions (Specific to this evening's trial) <br><br />
<table border="1"><br />
<tr><td> </td><td> Experimental Reaction </td> <td> Neg. Template Control </td> <td> Neg. Primer Control> </td></tr><br />
<tr><td> Template </td> <td> 2 uL </td> <td> 0 </td> <td> 2 uL </td></tr><br />
<tr><td>Forward Primer [VF2 310 uM] </td> <td> 16uL** </td> <td> 16uL** </td> <td> 0 </td></tr><br />
<tr><td>Reverse Primer [VR 345 uM] </td> <td> 14uL** </td> <td> 14uL** </td> <td> 0 </td></tr><br />
<tr><td> dNTPs </td> <td> 1uL </td> <td> 1uL </td> <td> 1uL </td></tr><br />
<tr><td> Rxn Buffer [5X-k] </td> <td> 10uL </td> <td> 10uL </td> <td> 10uL </td></tr><br />
<tr><td> Enzyme </td> <td> 0.5uL </td> <td> 0.5uL </td> <td> 0.5uL </td></tr><br />
<tr><td> Water </td> <td> 6.5uL </td> <td> 8.5uL </td> <td> 36.5uL </td></tr><br />
<tr><td> Total Volume </td> <td> 50uL </td> <td> 50uL </td> <td> 50uL </td></tr><br />
<tr><td> ** indicates 1:100 dilution </td></tr><br />
</table><br />
34mM of dried DNA in primers ($12.50) <br><br />
<br />
Several companies provide these primers: invitrogen is an example.<br><br />
<br />
34 x 10-9 moles / 100 x 10-6 liters = 3.4 x 10 -7 moles/liter<br><br />
<br />
Tomorrow we will look at the parts we pcr'd and then see how the bp length matches. If nothing happens its probably a pipetting error. If too many bands it may be an annealing issue.<br />
<br />
We will also look at the cells we plated of the various transformed parts, 3A, 5A, 7A, and see again if any of the colonies grew.<br />
<br />
----<br />
<br />
=== June 10, 2010 ===<br />
In attendance: Duke, Gary, Robert, Patrick, Ryan, Miles, Steven, Liz, and Colin.<br />
<br />
Tonight we tested the hypothesis that the plasmid did not code for the antibiotic resistance/that we did not use enough DNA. A Restriction Digest reaction was set up on the ligation reactions completed on June 7. The reactions were cut with EcoR1 and Pst1. The reactions were allowed to incubate at 37 degrees C for one and a half hours. The digests were then ran on a electrophoresis gel. No DNA was present as there was not enough DNA, thus proving Duke's hypothesis.<br />
<br />
----<br />
<br />
<br />
=== June 9, 2010 ===<br />
In attendance: Duke, Tom, Ryan, Patrick, Lisa, Steven.<br />
Missing: Kyle and Friend(Forgot his name)<br />
<br />
1) Registration for iGEM site & Team affiliation, required to update team wiki.<br />
<br />
2) Access and update wiki..<br />
Consider stylization, as well as information content. Who is our audience?<br />
Team info - blurb and caption of team members. Couple of sentences about you and your interests.<br />
Lab Notebook? - Openwetware connection.<br />
<br />
3) Research biobrick parts by accessing in Registry of Standard Parts.<br />
XF to see if new combination are already on file?<br />
If not begin documentation of the 3 new assemblies, with image using standard BioBrick icons.<br />
Document bp length of new parts to compare with Gel-Electrophoresis.<br />
How do we test efficacy? (Part for PPM still in brick.)<br />
<br />
Is the media okay, is the antibiotic right. Positive tells you whether the cells themselves were capable of taking up our DNA. (Ideally, they should use the same antibiotic resistance, as our parts.<br />
<br />
Negative groups tells us whether the antibiotic and media was effective and that the cells were not resistant to the media.<br />
<br />
We know that cells were competent they were able to be transformed, and the antibiotic was effective. So the remaining question is whether they were able to be transformed.<br />
<br />
The ligation's are in question. Generally the restrictions and ligation's go smoothly.<br />
<br />
Tom's Hypothesis is questioning whether the media plates may have been mis-labeled with the wrong antibiotic resistance. Since we used a different resistance for controls, there was no telling.<br />
<br />
Possible denaturing of enzymatic proteins from the heat shock on the initial restriction? 80c kill cycle.<br />
Vector ? - Linear plasmids - ''was the amount to small...?<br />
Parts - size already was confirmed, according to Tom<br />
<br />
No Transformations.... so now what? Primers still on the way. Don't need to go to scratch.<br />
<br />
<br />
The Core enzymes needed to perform the biobricks constructions...<br />
Spe1<br />
pst1<br />
ecor1<br />
xba1<br />
t4dna ligase<br />
polymerase<br />
<br />
Project idea: <br><br />
Polymerase in e.coli, tag, perhaps other enzymes could be self-manufactured/purified.<br />
<br />
Basic tools/measurements <br><br />
* Micropipetters [Measuring volume] <br><br />
* Measuring Mass <br><br />
* DV/HD camera - you tube videos and components. <br><br />
* Mini-preps. Growing the cells and isolating DNA. Cell competency Preparations. <br><br />
<br />
Lac polymerase - DIY-GEM<br />
<br />
How do you purify?<br />
<br />
What do you do with it... what is the process to create a project?<br />
<br />
Hardware only takes you so far, these 5 basic tools.<br />
Restrictions<br />
Ligations<br />
Transformations/Plating<br />
PCR<br />
Gel Electrophoresis<br />
<br />
The core will be good technique with these processes and then the ability to understand the existing database navigation with the proper questions to yield an experiment of interest.<br />
<br />
Research educational tools from the MIT educator on IGEM site.<br />
Should we have an e-mail/Comments section added to wiki - openwetware, so observers can ask us questions?<br />
<br />
<br />
----<br />
<br />
<br />
=== June 8, 2010 ===<br />
<br />
In attendence: Colin, Patrick, Liz, Ryan, Duke and Tom.<br />
Missing: Andy, Scott.<br />
<br />
So tonight we came in and took the 4 ligation's/new part combinations from yesterday and transformed them into the competent cells, using a heat-shock transformation, while also preparing the Control groups (one with nothing/one with the ampicillin resistance). 3 of the parts were Tetracycline resistant, and 1 was Chlorephenecol. After the 90 minute transformation cycle we plated 6 versions of each of the 3 parts along with 1 plate of control and one negative group.<br />
<br />
The 6 plates were done in a 0, -1, -2 dilutions in 2 concentrations one of 50 ul, and one of 200 ul.<br />
<br />
They will be left for tomorrows team to run gel electrophoresis to determine whether they have the appropriate combination links.<br />
<br />
<br />
----<br />
<br />
<br />
=== June 7, 2010 === <br />
In attendance: Colin, Patrick, Robert, Ryan, David, Gary, Miles, Duke and Tom.<br />
Missing: Melissa.<br />
<br />
Tom greeted us with 4 separate sheets that contained 3 reactions each for us to begin restrictions and began heating to let the enzymes cut. A lesson in what not to do, was offered as we began the heating cycle of restriction in the PCR blocks and he timing had been set to 35 seconds instead of 35 minutes, after which it heated to a kill cycle of 80 degrees and we had to reapply the enzymes, in case the enzymes had been denatured.<br />
<br />
We had 6 individuals building the 4 sheets, 2 in redundancy.<br />
<br />
The legend for the various parts is as follows...<br />
<table><br />
<tr><td>1PO</td><td><bbpart>BBa_R0063</bbpart></td></tr><br />
<tr><td>2PO</td><td><bbpart>BBa_P0412</bbpart></td></tr><br />
<tr><td>3PO</td><td><bbpart>pSBIT3</bbpart></td></tr><br />
<tr><td>R (black marker)</td><td><bbpart>BBa_R0062</bbpart></td></tr><br />
<tr><td>I (black marker)</td><td><bbpart>BBa_I13507</bbpart></td></tr><br />
<tr><td>S (black marker)</td><td><bbpart>pSBIT3</bbpart></td></tr><br />
<tr><td>F (green marker)</td><td><bbpart>BBa_F2620</bbpart></td></tr><br />
<tr><td>I (green marker)</td><td><bbpart><BBa_I13507</bbpart></td></tr><br />
<tr><td>S (green marker)</td><td><bbpart>SBIC3</bbpart></td></tr><br />
<tr><td>R10 (green marker)</td><td><bbpart>BBa_R0010</bbpart></td></tr><br />
<tr><td>462 (green marker)</td><td><bbpart>BBa_I0462</bbpart></td></tr><br />
<tr><td>IT3 (green marker)</td><td><bbpart>pSBIT3</bbpart></td></tr><br />
<tr><td>A/R10 (green marker)</td><td><bbpart>BBa_R0010</bbpart></td></tr><br />
<tr><td>B/462 (green marker)</td><td><bbpart>BBa_I0462</bbpart></td></tr><br />
<tr><td>C/IT3 (green marker)</td><td><bbpart>pSBIT3</bbpart></td></tr><br />
</table><br />
<br />
Patrick cut parts <bbpart>BBa_r0063</bbpart> and <bbpart>BBa_p0412</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>. <br><br />
Robert cut parts <bbpart>BBa_R0062</bbpart> and <bbpart>BBa_I13507</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>. <br><br />
Collin/Miles cut parts <bbpart>BBa_F2620</bbpart>, <bbpart>BBa_I13507</bbpart> and the plasmid backbone <bbpart>pSBIC3</bbpart> (labeled w/green marker). <br><br />
The next step is to ligate them.<br />
<br />
After which we had a round table discussion about what kind of projects we may follow up with and the process of using the NCBI databases to discover pre-existing sequence information related to our various ideas.<br />
One idea we have discussed was the option of creating a smoother introductory curve for fellow DIY-Bio commmunity members and the creation of home-brewed enzymes that might be to pricey for the amateur scientists.<br />
We ended the night with the beginning of the various ligation reactions, as seen above.<br />
<br />
*Ligation Reaction for R10+462+IT3 in 'LIGATE 05X''<br />
*Ligation Reaction for A/R10+B/462+C/IT3 in 'L/LIG''<br />
*Ligation Reaction for F2620+I13507+IC3 with "Green Asetrisk"<br />
*Ligation Reaction for R0062+I13507 in "Squiggly Sigil"<br />
<br />
|}</div>MPekalahttp://2010.igem.org/Team:Baltimore_US/JulyGroupNotebookTeam:Baltimore US/JulyGroupNotebook2010-10-27T05:29:07Z<p>MPekala: </p>
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{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
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!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
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!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
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{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
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<br><br />
== Group Notebook for July 2010 ==<br />
__TOC__<br />
<br />
=== July 29th, 2010 ===<br />
@ Roberts Lab / Towson <br />
Dr liz is wondering if anyone is bringing materials from CCBC over to the TU lab today. It is currently 1:50pm at the time of this post. Please email Dr Liz and let her know what, if anything, you are planning for today.<br><br />
<br />
<br />
<br />
<br><br />
=== July 28th, 2010 ===<br />
@ Roberts Lab / Towson<br />
In Attendance: Dr Roland Roberts, Dr Liz, Dr Schiefele, Patrick, Ryan, Duke <br><br />
<br><br />
Dr Liz introduced this group of iGEM folks to Dr Roland Roberts. Dr Roberts is willing to have a small cadre of the iGEM Team Baltimore work in his lab. Because Dr Roberts is not officially an iGEM Advisor, we are going to be especially considerate about using his lab space. '''We are not going to have any more people come into his lab space at this time.''' There may be room for one more person to work in the Roberts' Lab at a later date.<br><br />
<br><br />
Dr Roberts has a very disctinct protocol in his lab, and he was kind enough to distribute Lab Instructions to all of us -- these are what he gives his own students when they first comoe into his lab. We are very grateful for Dr Roberts' support, and we will show that by being especially sensitive to his lab policies and procedures.<br />
Dr Roberts keeps a sterile working environment. PCR reactions and cloning are always done under the hood. His lab only uses filtered tips. Dishes do not stack up in his sink. We will help do the autoclaving. Ethidium Bromide is done in only one area of the lab - it is stained into gels after they are run, rather than put into the gel agarose. The reasons for all of Dr Roberts' protocols are completely obvious. (Even if they weren't, we would follow them anyway!) <br><br />
<br><br />
We will have to provide ALL of our expendables - from gloves, tape, felt-tips, foil for autoclaving, tissue wipes, clips and storage bottles, to all reagents we need for reactions. That means gel agarose, molecular ladder, restriction enzymes, ligase, competent cells, antibiotics, cell culture plates and agar, DNA oligos, primers, dntp's, polymerase, BSA, buffers for PCR and Restriction digestion reactions. We also need to bring over a gel apparatus or two (he has ample power supplies), pipetmen, filter tips, tubes of all sizes, work blocks for holding tubes, chemical jars (orange lids) if we need to mix up own own solutions. Other needs will become apparent as we begin to perform experiments in the Roberts lab.<br><br />
<br><br />
We do not have to provide our own TAE, TBA or paper towels. These items are cheap enough that Dr Roberts can let us use his. We will not bring ethidium bromide for now. Maybe later.<br><br />
<br><br />
We may use Dr Roberts' glassware. If you break it, Dr Liz buys it. Please be careful! We have access to PCR machines, rockers, water bath, centrifuges, spinners, cold centrifuge, fridge, freezers (both -20 and -80), microwave, autoclave, and the gel imaging system. The cell culture room is on the 4th floor of Smith and is open all the time for anyone's use.<br><br />
<br><br />
We may also use the PC's in the lab for lab-related work or checking email. (Please do not update your facebook account on Dr Roberts' PC's!). He has two pipet stands - one has been spoken for by Dr Liz. Sr Jim Saunders, Director of the MB3 Program, has gotten lengthy wish lists over the last few days from Dr Liz, and he has already provided storage boxes for tubes. He emailed Dr liz yesterday and said he has more stuff. Dr Saunders is sure to be a great resource. <br><br />
<br><br />
In Dr Robert's lab, it goes without saying that Dr Roberts' say is final. Otherwise, Dr Liz's say is final. It is her reputation on the line at the Towson Lab space, and anything the iGEM team does will reflect directly on Dr Liz. Anyone who cannot work under that caveat should bow out now from the Towson Lab space. <br><br />
<br><br />
On a more upbeat note, the Towson Lab will have results to report soon, we hope!<br><br />
<br><br />
<br />
=== July 27th, 2010 ===<br />
@ Burkett Lab <br />
Day Shift: Patrick, Robert, Duke and Ryan<br><br />
<br><br />
Running restriction digests on ANN parts for validation. Problem with <partinfo>J23030</partinfo>: restriction digest shows only a faint band of genomic DNA in lanes 2 & 3 (cut, uncut with Spe1):<br />
<br />
http://imgur.com/ePNEO.jpg<br />
<br />
Will run a larger gel tomorrow with all ANN parts to confirm.<br />
<br />
--[[User:Pon|Pon]] 22:40, 27 July 2010 (UTC)<br />
<br />
Ryan ran the transformations for the PoliColi Ignition Ligation. (<partinfo>R0010</partinfo>, <partinfo>B0034</partinfo>, <partinfo>PsB1C3</partinfo>)<br><br />
https://static.igem.org/mediawiki/2010/c/c2/BbPart_%28Ignition-R0010%2CB0034%2CPsB1C3%29Transformation.pdf<br><br />
When we return to the lab on thursday, cell plates must be checked from incubator. Looking especially for results on RXN plate, sample should be run for electrophoresis sizing after additional culturing.<br><br />
<br><br />
<br />
=== July 26th, 2010 ===<br />
- Burkett Lab <br />
Attendence: Robert, Duke, Ryan, Patrick<br><br />
<br><br />
Ryan ran the Photospectrometer analysis on the Bb DNA that's been cultured for the ANN and PolIColi ignition components.<br />
Results are as follows...<br><br />
<br><br />
<bbpart>J01001</bbpart> A260 = 1.03 Concentration= 51.57 ug/ml Concentration = 0.05 ug/ul<br><br />
<bbpart>J01003</bbpart> A260 = 2.37 Concentration = 118.55 ug/ml Concentration = 0.12 ug/ul<br><br />
<bbpart>J23007</bbpart> A260 = 1.33 Concentration = 66.72 ug/ml Concentration = 0.07 ug/ul<br><br />
<bbpart>J23008</bbpart> A260 = 2.52 Concentration = 125.74 ug/ml Concentration = 0.13 ug/ul<br><br />
<bbpart>J23022</bbpart> A260 = 1.87 Concentration = 93.46 ug/ml Concentration= 0.09 ug/ul <br><br />
<bbpart>J23030</bbpart> A260 = 3.11 Concentration = 155.94 ug/ml Concentration = 0.16 ug/ul<br><br />
<bbpart>J23031</bbpart> A260 = 0.14 Concentration = 7.11 ug/ml Concentration = 0.01 ug/ul<br><br />
<bbpart>J23032</bbpart> A260 = 2.01 Concentration = 100.36 ug/ml Concentration = 0.10 ug/ul<br><br />
<bbpart>J23036</bbpart> A260 = 2.08 Concentration = 104.07 ug/ml Concentration = 0.10 ug/ul<br><br />
<br><br />
<bbpart>R0010</bbpart> A260 = 1.44 Concentration = 72.19 ug/ml Concentration = 0.07 ug/ul<br><br />
<bbpart>B0034</bbpart> A260 = 2.55 Concentration = 127.36 ug/ml Concentration = 0.13 ug/ul<br><br />
<bbpart>B0015</bbpart> A260 = 2.43 Concentration = 121.23 ug/ml Concentration = 0.12 ug/ul<br><br />
<br><br />
<br />
=== July 23rd, 2010 ===<br />
at Towson Campus <br />
In Attendance: Liz, Roland Roberts, Jim Saunders<br><br />
<br><br />
Dr Liz met with Dr Roberts and was given the tour of the lab space and equipment available for Team Baltimore to use if a subgroup wants to work at TU. All the usual equipment is available except Dr Roberts want Team Baltimore at TU to bring their own set of pipetment, and of course, supply its own needs for expendable items. Dr Jim Saunders has taken a copy of Team Baltimore at TU's wish list, and says he can provide a set of pipetment, and tips/tubes without much trouble. We also need to take a gel electrophoresis apparatus, because Dr Roberts has a limited supply and other students use them frequently. He does have an extremely large one he said we could use.<br><br />
Dr Roberts wants to meet with the students who wish to have access to his lab, sooner rather than later. It is important that Team Baltimore bring reagents over to the Roberts Lab and begin to use some of the space Dr Roberts has set aside for us, in order to demonstrate that we are serious about using his lab. Anyone who would like to work at the TU site sometimes should contact Dr Liz immediately.<br><br />
There is a DNA sequencer and a high-throughput DNA seperator that is accurate to 1 bp for oligos between 100-650 bp long. If Team Baltimore wants to use this machine, that is possible. In that case Dr liz will talk to her Dean at TU to acquire funds to help share the cost of using it.<br><br />
<br />
<br><br />
<br />
=== July 20th, 2010 ===<br />
In Attendance: Gary, Bernadette, Tom, Patrick, Ryan<br><br />
During day shift, Robert finished working on PCR optimizations. While Patrick and Ryan, troubleshot their primers and put the order in so that they can begin PCR reactions when they return on monday.<br><br />
Bernadette concentrated on her Ligation reactions for the PolIColi project. <br><br />
Ligation reaction (COLOR CODED RED)included: 6 ul R0010 promoter, 6 ul B0034 RBS, <br><br />
2 ul PsB1C3 chloremphenacol resistant plasmid,2 ul buffer, 3 ul H20 & 1 ul ligase enzyme <br><br />
For comparison, I also completed a 'No Insert' group (COLOR CODED TEAL/BLUE)using 2 ul plasmid, 2 ul buffer,<br> <br />
15 ul H20, 1 ul ligase<br><br />
In addition a 'No Enzyme' group (COLOR CODED DARK GREEN)was completed that included 6 ul R0010 promoter, <br><br />
6 ul B0034 RBS, 2 ul plasmid, 2 ul buffer, 4 ul H20 <br><br />
The 3 groups were incubated at 16 degrees C overnight<br />
<br />
<br />
<br><br />
Gary got caught up on project status and development.<br><br />
<br><br />
<br />
=== July 19th, 2010 ===<br />
In attendance: Miles, Patrick, Robert, Duke, Liz, Tom, Bernadette, Steven<br><br />
Steve transformed <partinfo>J23030</partinfo> as it was the only DNA from last week's transformations that did not develop any colonies.<br><br />
Bernadette began restriction digestions for the Bbparts for the PoliColi project.<br><br />
Part #'s<br><br />
<partinfo>R0010</partinfo> The LacI Promoter<br><br />
<partinfo>B0034</partinfo> The Strong RBS<br><br />
<partinfo>PsB1C3</partinfo> A Chloremphenacol Resistant Plasmid Backbone<br><br />
<partinfo>B0015</partinfo> The Double Terminator<br><br />
<partinfo>R0010</partinfo> and <partinfo>B0034</partinfo> are meant to be ligate into the <partinfo>PsB1C3</partinfo> as all the initial PoliColi Bbparts had a native Amp resistance and one had Amp and Kanamyacin.<br><br />
The <partinfo>B0014</partinfo> will be ligated with the PoliColi NewPart once the Overlap Extension Point Mutation PCR is completed.<br><br />
<br />
Restriction digestion included the following: R0010 Promoter (COLOR CODED BLUE): 5 ul DNA, 5 ul buffer#4, <br><br />
37.5 ul H20,cuts made with 1 ul EcoRI and 1 ul SpeI enzymes, and .5 ul BSA The result will be the insert <br><br />
In a separate reaction B0034 RBS (COLOR CODED PURPLE): 5 ul DNA, 5 ul buffer #4, 37.5 ul H20, 1 ul XbaI,<br><br />
1 ul PstI,.5 ul BSA <br><br />
The 3rd reaction PsB1C3 plasmid(COLOR CODED LIGHT GREEN): 5 ul DNA, 38 ul H20, 5 ul buffer #4, 1 ul EcoRI,<br> <br />
1 ul PstI, no BSA <br><br />
Incubated overnight 37 degrees C<br />
<br />
Ryan and Patrick began working on Primer design for their various PCR projects.<br><br />
Miles showed off initial development of Gel Electrophoresis Kit and Power Supply. Discussed PCR construction with Tom, as well as introduction to Oligonucleotide synthesis.<br><br />
<br><br />
<br />
=== July 12th - 15th, 2010 ===<br />
Dayshift Lab Techs began DNA Preparation of various identified BbParts. Robert and Duke finished with Boiling Prep.<br><br />
Evening Lab intermittently closed with Tom unavailable for lab supervision.<br><br />
<br><br />
<br />
=== July 8th, 2010 ===<br />
In Attendance: Gary, Patrick, Robert, Lisa, Liz, Steven<br />
Strategy Meeting in spare room. Patrick outlined strategy of ANN (Artificial Neural Network) based on Berkeley's previous work. Discussed use of conjugation plasmids for weight assessment.<br><br />
<br><br />
<br />
=== July 6th, 2010 ===<br />
Can't quite remember back far enough to state what occurred. Got to remind everyone to update the notebook wiki page as during the evening's in question.<br><br />
<br><br />
<br />
=== July 1st, 2010 ===<br />
Ryan didn't make it, but I hear something happened.<br />
<br><br />
<br />
|}</div>MPekala