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	===Primer Design===		===Primer Design===
		+	SO1415
		+
		+	· Gene sequence: ‘’’Overhang’’’ of the actual gene
		+	· This sequence was found on http://www.ncbi.nlm.nih.gov/
		+	· Megablast was used to compare the designed primer against the s.
		+	oneidensis genome
		+	· the length, gc content, melt temp etc info was found on www.idtdna.com
		+	· the site used to find parameters of a well-designed primer:
		+	http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
		+
		+	SO_1415 gene sequence:
		+	5’
		+	‘’’GAATGAATAA’’’
		+	ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA
		+	TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC
		+	TTTATATAAATTTTTTGAACGTAAAGAAGATGTGTTGGTTTGTTTATTTTTAAGAAGCGCAACCTCAAAT
		+	CACTTACCGATATTTATCCATAAAAATCCAGAGTTAACTGCGCAAGAGAAGGTGCTGTTACCCATTTTAT
		+	TTACCTTTGAAACCATTAAGCGCAGTAGTAGCTTTTTTACGCTGCGTTCGGTGTCGGTCAATACCATGGT
		+	GTGGAAACTGGCCAGTGACGAAAAAGTGGAGCGGTTTAAAAAACGCATTAATGCTTTTTGGAGTTGGTTT
		+	ACAGACTCACTGCATTTAGCTGTTGAAAACGGCGAATTAGTGGCAACACCATTACAAATTAAAGAATTGG
		+	TCCAAGGGATAACGTTTTATTTAACAGGGTCTTTGACACAATTTGAAAGTCAATTGATTGCCCCAGAGTT
		+	TTTGTCTGATCGCCGTGAAACCTGTTATCGACATTTAGCAAACCTGATGGAGCGATACGAGTGGAAAAAG
		+	CCTTTAACTCTTGCGCTGTTTGATTCGTTAGAGGCGAGAACTATTAAGTTTTTTGACCAACATTATCGTG
		+	ACCATATGACCTGCGCGGCTTGTAGTGCGCTGTCAAATACCGACACTAAGACATCATCTCCCTGTACTCG
		+	TCAGTGTGGTTAG
		+	‘’’GGCGTCCTGC’’’
		+	3’
		+
		+	Primer 1:
		+	‘’’TGA ATA A’’’AT GAA ATG TCC TTC GGA CTC CCT G
		+	LENGTH:31
		+	GC CONTENT:41.9 %
		+	MELT TEMP:59.9 ºC
		+	MOLECULAR WEIGHT:9494.2 g/mole
		+	EXTINCTION COEFFICIENT:298500 L/(mole·cm)
		+	nmole/OD260:3.35
		+	µg/OD260:31.81
		+
		+	Primer 2:
		+	‘’’GAC GCC’’’ CTA ACC ACA CTG ACG
		+	LENGTH:21
		+	GC CONTENT:61.9 %
		+	MELT TEMP:60.2 ºC
		+	MOLECULAR WEIGHT:6345.2 g/mole
		+	EXTINCTION COEFFICIENT:197000 L/(mole·cm)
		+	nmole/OD260:5.08
		+	µg/OD260:32.21
		+
		+
		+
		+	SO4157
		+
		+	5’
		+	‘’’AAGGAAAACC’’’
		+	ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC
		+	TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC
		+	ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA
		+	CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG
		+	CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA
		+	AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC
		+	GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT
		+	ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC
		+	AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA
		+	‘’’TCCAATAAAC’’’
		+	3’
		+
		+	Primer 1:
		+	‘’’AAA ACC’’’ ATG TCC ACC ATG CTG C
		+	LENGTH:22
		+	GC CONTENT:50.0 %
		+	MELT TEMP:58.7 ºC
		+	MOLECULAR WEIGHT:6648.4 g/mole
		+	EXTINCTION COEFFICIENT:207700 L/(mole·cm)
		+	nmole/OD260:4.81
		+	µg/OD260:32.01
		+
		+	Primer 2:
		+	‘’’ATT GGA’’’ TTA CTC CGG TAA GAG TGT ATT TAG GT
		+	LENGTH:32
		+	GC CONTENT:37.5 %
		+	MELT TEMP:58.6 ºC
		+	MOLECULAR WEIGHT:9924.5 g/mole
		+	EXTINCTION COEFFICIENT:320800 L/(mole·cm)
		+	nmole/OD260:3.12
		+	µg/OD260:30.94
		+
		+
		+
		+
		+
		+
		+
		+
		+
		+
		+
		+
		+
		+	hlyU
		+
		+	‘’’ATGAAAACCA’’’
		+	TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT
		+	ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG
		+	CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA
		+	AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC
		+	ATCAATTCAATATTCAT
		+	‘’’TACGCACTTT’’’
		+
		+	Primer 1:
		+	‘’’ATG AAA ACC A’’’TT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG
		+	LENGTH:41
		+	GC CONTENT:22.0 %
		+	MELT TEMP:56.6 ºC
		+	MOLECULAR WEIGHT:12655.3 g/mole
		+	EXTINCTION COEFFICIENT:432600 L/(mole·cm)
		+	nmole/OD260:2.31
		+	µg/OD260:29.25
		+
		+	Primer 2:
		+	‘’’GTG CGT A’’’AT GAA TAT TGA ATT GAT GCA GGA
		+	LENGTH:30
		+	GC CONTENT:36.7 %
		+	MELT TEMP:57.8 ºC
		+	MOLECULAR WEIGHT:9349.1 g/mole
		+	EXTINCTION COEFFICIENT:309700 L/(mole·cm)
		+	nmole/OD260:3.23
		+	µg/OD260:30.19
	== Week's (Ambitious) Goals ==		== Week's (Ambitious) Goals ==

---

Revision as of 20:36, 31 May 2007

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Contents 1 What We've Accomplished 1.1 Primer Design 2 Week's (Ambitious) Goals 3 Materials We Need 4 Short-Term To-Do List 5 Relevant Publications and Links

What We've Accomplished

Primer Design

SO1415

· Gene sequence: ‘’’Overhang’’’ of the actual gene · This sequence was found on http://www.ncbi.nlm.nih.gov/ · Megablast was used to compare the designed primer against the s. oneidensis genome · the length, gc content, melt temp etc info was found on www.idtdna.com · the site used to find parameters of a well-designed primer:

     http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

SO_1415 gene sequence: 5’ ‘’’GAATGAATAA’’’ ATGAAATGTCCTTCGGACTCCCTGTCCATTTTACGTTGTAATCAAATATTGGATGCGGCTGAAAAGCTCA TTGAGTCACAAGGTGTTGTATCTTTTAAGTTTTCTCAGCTTGCGCATGAGGTGGGATGCTCTACGGGTAC TTTATATAAATTTTTTGAACGTAAAGAAGATGTGTTGGTTTGTTTATTTTTAAGAAGCGCAACCTCAAAT CACTTACCGATATTTATCCATAAAAATCCAGAGTTAACTGCGCAAGAGAAGGTGCTGTTACCCATTTTAT TTACCTTTGAAACCATTAAGCGCAGTAGTAGCTTTTTTACGCTGCGTTCGGTGTCGGTCAATACCATGGT GTGGAAACTGGCCAGTGACGAAAAAGTGGAGCGGTTTAAAAAACGCATTAATGCTTTTTGGAGTTGGTTT ACAGACTCACTGCATTTAGCTGTTGAAAACGGCGAATTAGTGGCAACACCATTACAAATTAAAGAATTGG TCCAAGGGATAACGTTTTATTTAACAGGGTCTTTGACACAATTTGAAAGTCAATTGATTGCCCCAGAGTT TTTGTCTGATCGCCGTGAAACCTGTTATCGACATTTAGCAAACCTGATGGAGCGATACGAGTGGAAAAAG CCTTTAACTCTTGCGCTGTTTGATTCGTTAGAGGCGAGAACTATTAAGTTTTTTGACCAACATTATCGTG ACCATATGACCTGCGCGGCTTGTAGTGCGCTGTCAAATACCGACACTAAGACATCATCTCCCTGTACTCG TCAGTGTGGTTAG ‘’’GGCGTCCTGC’’’ 3’

Primer 1: ‘’’TGA ATA A’’’AT GAA ATG TCC TTC GGA CTC CCT G LENGTH:31 GC CONTENT:41.9 % MELT TEMP:59.9 ºC MOLECULAR WEIGHT:9494.2 g/mole EXTINCTION COEFFICIENT:298500 L/(mole·cm) nmole/OD260:3.35 µg/OD260:31.81

Primer 2: ‘’’GAC GCC’’’ CTA ACC ACA CTG ACG LENGTH:21 GC CONTENT:61.9 % MELT TEMP:60.2 ºC MOLECULAR WEIGHT:6345.2 g/mole EXTINCTION COEFFICIENT:197000 L/(mole·cm) nmole/OD260:5.08 µg/OD260:32.21

SO4157

5’ ‘’’AAGGAAAACC’’’ ATGTCCACCATGCTGCCACTGTATTTAGTCGATGATGATGAAGCGATTCTCGACTCCTTAGGGTTTATGC TCAGGCAATTTGGTTACCAAGTACAAACCTTTAGCAGTGGACGGGATTTTTTAGCCCAATGTCCGTTAAC ACAGGCTGGCTGCGTGATTTTAGATAGCCGAATGCCGGAGATCACCGGCCAAGAAGTGCAGCAAAAACTA CTTGAAACCCAAAGCCCATTGGGAGTTATCTTTCTCACGGGGCACGGTGATTTGCCCATGGCATTAAGCG CCTTTCGTAAGGGTGCATGCGATTTTTTTCAAAAGCCGGTATCTGGCAAAGCCCTAGTACAAGCCATTAA AAAAGCGCATAAAGAAAGCCAAGCCAGCTTTGAGCAACAGAGTCTGCAGCATAAATTTGCCCAACTGACC GACCGTGAACAACAAGTGTTAGCCCATGTGGTTCAAGGTATGACCAACAAGCAGATCTCCGAGGCCATGT ATTTATCCTTAAGAACCATTGAAGTGCACCGCGCTAAGATCATGAAAAAGCTCGAAGTCAGTAATATGGC AGAATTAGTACAGCACTTAGCCCACCTAAATACACTCTTACCGGAGTAA ‘’’TCCAATAAAC’’’ 3’

Primer 1: ‘’’AAA ACC’’’ ATG TCC ACC ATG CTG C LENGTH:22 GC CONTENT:50.0 % MELT TEMP:58.7 ºC MOLECULAR WEIGHT:6648.4 g/mole EXTINCTION COEFFICIENT:207700 L/(mole·cm) nmole/OD260:4.81 µg/OD260:32.01

Primer 2: ‘’’ATT GGA’’’ TTA CTC CGG TAA GAG TGT ATT TAG GT LENGTH:32 GC CONTENT:37.5 % MELT TEMP:58.6 ºC MOLECULAR WEIGHT:9924.5 g/mole EXTINCTION COEFFICIENT:320800 L/(mole·cm) nmole/OD260:3.12 µg/OD260:30.94

hlyU

‘’’ATGAAAACCA’’’ TTAATGACAATAAATATTGTTCAATAAATGGATCATCTCACGTACCTCATCACTTTTCAGTGAGTAGAAT ACAGTTTGCGCTTCTTTGCGTGTGGTCACTAAATTATCTTTGCGCAACCAAGCAAGGTGTTGTGATAGTG CCGATTGACTTAAGCCTAATTTTTTATTCATTTCGCCAACGCACATTTCTCCTTCATTCAATAAATAACA AAGGATAAATAAACGGCGTTCGTTTGCGAGTGCCTTTAATAGCACCACGGCATGATCGGCTCGCTCCTGC ATCAATTCAATATTCAT ‘’’TACGCACTTT’’’

Primer 1: ‘’’ATG AAA ACC A’’’TT AAT GAC AAT AAA TAT TGT TCA ATA AAT GG LENGTH:41 GC CONTENT:22.0 % MELT TEMP:56.6 ºC MOLECULAR WEIGHT:12655.3 g/mole EXTINCTION COEFFICIENT:432600 L/(mole·cm) nmole/OD260:2.31 µg/OD260:29.25

Primer 2: ‘’’GTG CGT A’’’AT GAA TAT TGA ATT GAT GCA GGA LENGTH:30 GC CONTENT:36.7 % MELT TEMP:57.8 ºC MOLECULAR WEIGHT:9349.1 g/mole EXTINCTION COEFFICIENT:309700 L/(mole·cm) nmole/OD260:3.23 µg/OD260:30.19

Week's (Ambitious) Goals

Wednesday 5/30

1. Get all protocols
2. Identify materials/prepare order
3. Design Primers
4. Learn about budget/POs

Thursday 5/31

1. Do primer order
2. Start conjugation practice
3. Confirm restriction enzymes, ligases
4. Order confirmed/needed materials
5. Team Revew Meeting
6. Draft Thank-You Letters for our Sponsors

Friday 6/1

1. Evaluate/continue conjugation, practice electroporation for E. coli
2. Revise proposal to include possibility of screening with alginate beads and fluorocytometer
3. Meeting with Tim: Budgets/protocols, Pfizer/fundraising, iGEM registration, beads

Materials We Need

Primers: Need to Buy

Error-Prone PCR: Need to Buy

Plasmids: Need to Buy?

Restriction Enzymes: Need to Buy?

Ligases: Need to Buy?

Short-Term To-Do List

Lab Orientation: COMPLETED!

• Lab Orientation Checklist

Lab Safety Training: Not Completed (Dave, Rahul, and Christian are scheduled for a training session on 5/30/07. Danny will be taking it next week)

Design of Primers: Not Completed (Dave, please send me info about the finished primers when ready)

Ordering of Primers: Not Completed

Gathering of Protocols: Not Completed (Chris, please send me the protocols when they are gathered)

Ordering of Error-Prone PCR Materials: Not completed

Thank-You Letters sent to Pfizer: COMPLETED!

Thank-You Letters sent to BU ppl: Not completed

Relevant Publications and Links

http://www.shewybase.bu.edu

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