Team:Baltimore US/Project
From 2010.igem.org
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PolI<br> | PolI<br> | ||
J04639.1<br> | J04639.1<br> | ||
- | + | [Sequence http://www.ncbi.nlm.nih.gov/nuccore/155128] | |
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- | + | We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA. [http://research.med.helsinki.fi/plasmadna/ Plasma DNA] is free software from University of Helsinki which provides quick analysis of plasmid sequence information. | |
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- | We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA. Plasma DNA is | + | |
<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> | 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> | ||
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CTGCAG-PstI restriction site<br> | CTGCAG-PstI restriction site<br> | ||
GACGTC-Complement<br> | GACGTC-Complement<br> | ||
- | Solution - Site-specific Mutagenesis by Overlap Extension (see | + | Solution - Site-specific Mutagenesis by Overlap Extension (see [http://www.cshlpress.com/default.tpl?cart=1279686078181232350&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=21 Sambrook, Joseph; Russell, David W. ; Molecular Cloning: A Laboratory Manual, 3rd Edition]) |
<br><br> | <br><br> | ||
- | We then used the Gene Designer 2.0 | + | We then used the Gene Designer 2.0 from [https://www.dna20.com/genedesigner2/ DNA2.0] to analyze the open reading frames and examine the codons within the PstI restriction site. We find that the first three are coding for leucine with CTG and can be changed at one point to CTT without sacrificing functional integrity in the manufactured enzyme.<br> |
====Primer Design==== | ====Primer Design==== | ||
- | We designed | + | We designed a primer pair in order to induce point-mutagenesis at the Pst1 restriction site, flanking the base pair to be altered by 14 nt. 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> |
GTGGAGAAGATCCT(T)CAGTACCGGCGG<br> | GTGGAGAAGATCCT(T)CAGTACCGGCGG<br> | ||
CACCTCTTCTAGGA(A)GTCATGGCCGCC<br> | CACCTCTTCTAGGA(A)GTCATGGCCGCC<br> | ||
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'''Voila!!!''' Brand New Taq Polymerase Bb Part.<br> | '''Voila!!!''' Brand New Taq Polymerase Bb Part.<br> | ||
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== Developing low-cost alternatives to existing hardware: Project Details and Results == | == Developing low-cost alternatives to existing hardware: Project Details and Results == |
Revision as of 02:34, 28 October 2010
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DIY-GEM: a path towards low cost high throughput gene synthesis.Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler. Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects." Developing low-cost alternatives to existing enzymes: Taq polymerase Project DetailsThermus Aquaticus Polymerase I Problem: PstI restriction site - Found @ 1717CTGCAG-PstI restriction site Primer DesignWe designed a primer pair in order to induce point-mutagenesis at the Pst1 restriction site, flanking the base pair to be altered by 14 nt. with changed Amino Acid Bp's Targeting initial Leucine at G of CTG to CTT. Point mutation Original G in CTG of Leucine. Change of one base to CTT maintains Leucine integrity. GTGGAGAAGATCCT(T)CAGTACCGGCGG 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) PolI Coli Primers For Overlap Extension PCRPCR Reaction 1
Developing low-cost alternatives to existing hardware: Project Details and ResultsAn 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.
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