Team:Baltimore US/Project

DIY-GEM: a path towards low cost high throughput gene synthesis.

Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler. Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects."

PoliColi Project Details

Thermus Aquaticus Polymerase I
PolI
J04639.1
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128

    1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC

   51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG

  101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC

  151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT

  201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG

  251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC

  301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC

  351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC

  401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC

  451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA

  501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG

  551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG

  601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA

  651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC

  701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG

  751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC

  801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG

  851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA

  901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA

  951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC

 1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG

 1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC

 1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG

 1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC

 1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC

 1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT

 1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG

 1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT

 1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA

 1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC

 1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT

 1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC

 1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC

 1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC

 1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA

 1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC

 1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC

 1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA

 1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC

 1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA

 2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA

 2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG

 2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG

 2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG

 2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC

 2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG

 2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG

 2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC

 2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC

 2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG

 2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG

 2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG

 2601 GCTCTCCGCC AAGGAGTGAT ACCACC

We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA. Plasma DNA is freeware from University of Helsinki which provides quick analysis of plasmid sequence information. http://research.med.helsinki.fi/plasmadna/
When we cut and paste this dna sequence into plasmadna and look at the output window, we are given a visual output of various coding information. Such as restriction sites found within the code. To consider a construct viable for a BbPart we'll need to make certain that the standard restriction enzymes used with the system won't sheer the dna making it incomplete code. Searching for EcoRI, Xbe1, Sbe1, Pst1 sites will show whether the code is viable in an untampered state.

Problem: PstI restriction site - Found @ 1717

CTGCAG-PstI restriction site
GACGTC-Complement
Solution - Site-specific Mutagenesis by Overlap Extension (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)

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.

Primer Design

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.

GTGGAGAAGATCCT(T)CAGTACCGGCGG

CACCTCTTCTAGGA(A)GTCATGGCCGCC

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)

Important considerations are Melting Point and percentage CG complements. Other considerations are dimerizations, that might cause primers to hairpin.
We analyzed these primers using the OligoAnalyzer at IDT. When analyzing PolI Complements only were used for sequence inquiry, not the Bb Suffix/Prefixes.
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

PolI Coli Primers For Overlap Extension PCR

PCR Reaction - 1

Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC
60.5 c ; 56.5 % GC Concetration

TAQ Rm
CTCCCGGTACTGAAGGATCTTCTCCAC
61.5 c ; 55.6 % GC Concentration

PCR Reaction - 2

TAQ Fm
GTGGAGAAGATCCTTCAGTACCGGGAG
61.5 c; 55.6 % GC

Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC
61.8 c; 65 % GC

PCR Reaction - 3
Bb Prefix & Suffix Primers

Resuspend in 100 uL of H2O
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)

NEXT
- Create Full Bb Prmr w Plasmid combining new part using

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

<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS

Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI
Cut <partinfo>B0034</partinfo> w/XbeI & PstI

Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because

---
New Part + <partinfo>B0015</partinfo> = New Part

Cut New Part w/EcoRI & SpeI
Cut <partinfo>B0015</partinfo> w/XbeI & PstI

Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)

Cut 1st Combined Part w/EcoRI & SpeI
Cut 2nd Combined Part w/XbeI & PstI

Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)

Voila!!! Brand New Taq Polymerase Bb Part.

Part 2

The Experiments

We began the first few weeks meeting to orient ourselves with the structure of the Registry.

Part 3

Results