Assembly
From 2006.igem.org
(→'''Building''') |
(→'''hix BioBricks''') |
||
Line 66: | Line 66: | ||
HixL | HixL | ||
- | + | GAATTC GCGGCCGC T TCTAGA TTCTTGAAAACCAAGGTTTTTGATAA ACTAGT A GCGGCCGC CTGCAG | |
HixR | HixR | ||
- | + | GAATTC GCGGCCGC T TCTAGA TTATCAAAAACCTTCCAAAAGGAAAA ACTAGT A GCGGCCGC CTGCAG | |
HixC | HixC | ||
- | + | GAATTC GCGGCCGC T TCTAGA TTATCAAAAACCATGGTTTTTGATAA ACTAGT A GCGGCCGC CTGCAG | |
'''Oligos to order''' | '''Oligos to order''' | ||
Line 80: | Line 80: | ||
HixL_top | HixL_top | ||
- | + | GAATTCGCGGCCGCTTCTAGATTCTTGAAAACCAAGGTTTTTGATAAACTAGTAGCGGCCGCCTGCAG | |
HixL_bottom | HixL_bottom | ||
- | + | CTGCAGGCGGCCGCTACTAGTTTATCAAAAACCTTGGTTTTCAAGAATCTAGAAGCGGCCGCGAATTC | |
HixR_top | HixR_top | ||
- | + | GAATTCGCGGCCGCTTCTAGATTATCAAAAACCTTCCAAAAGGAAAAACTAGTAGCGGCCGCCTGCAG | |
HixR_bottom | HixR_bottom | ||
- | + | CTGCAGGCGGCCGCTACTAGTTTTTCCTTTTGGAAGGTTTTTGATAATCTAGAAGCGGCCGCGAATTC | |
HixC_top | HixC_top | ||
- | + | GAATTCGCGGCCGCTTCTAGATTATCAAAAACCATGGTTTTTGATAAACTAGTAGCGGCCGCCTGCAG | |
HixC_bottom | HixC_bottom | ||
- | + | CTGCAGGCGGCCGCTACTAGTTTATCAAAAACCATGGTTTTTGATAATCTAGAAGCGGCCGCGAATTC | |
== '''Building''' == | == '''Building''' == |
Revision as of 14:41, 2 June 2006
Parts to Make:
HixL: TTCTGGAAAA CCAAGGTTTT TGATAA
HixR: TTATCAAAAA CCTTCCAAAA GGAAAA
HixC: TTATCAAAAA CCATGGTTTT TGATAA
Backwards Parts
Because we wish to start with elements that are reversed (upside down pancakes), we need to think about how to get backwards parts. That is, parts that have the Prefix, then reversed element (promoter, term, CDS, RBS), then Suffix. Several possible ways:
- Some parts are backwards in the registry - terminators, for eg.
- Parts that are not long could be synthesized backwards.
- If one pancake can be flipped, reversed parts can be made. This may actually be a good contribution, since it could be applied to reverse any part between two hix sites.
Examples
BBa_B0022 Terminator (Reverse B0012) Length: 84 Base Pairs Including BioBrick Ends
Bingo! BBa_P1004 is ampR resistance cassette in reverse orientation Length: 984 Base Pairs Including BioBrick Ends
Bba_B0034 is a RBS with the sequence:
gaattcgcggccgcttctagag aaagaggagaaa tactagtagcggccgctgcag
It could be redesigned with the RBS reversed:
gaattcgcggccgcttctagag tttctcctcttt tactagtagcggccgctgcag
Steps
1. Design/Order DNA – Hix L, R, & C
- Want to do a Left to Right BioB addition: Promoter + RBS w/AmpR -->Hix site--> Term-->Hix(C)site-->TetR + Term
2. On receiving – restrict/ligate BioBricks into plasmid w/resistance gene on one side
a. Promoter, RBS BioB
b. TetR BioB (Backwards)
c. Term BioB
3. Transform E. coli
E = G/AATC
X = T/CTAGA
P = CGAT/CG
S = A/CTAGT
hix BioBricks
Hey, it's your Davidson pals.We noticed that your biobricks do not have the Not1 site between E and X in the prefix and S and P in the suffix Thanks.
E - N - X - hix - S - N - P
Does there have to be a T between NotI and XbaI and an A between SpeI and NotI? All the parts we looked at (10 of them) had this.
HixL
GAATTC GCGGCCGC T TCTAGA TTCTTGAAAACCAAGGTTTTTGATAA ACTAGT A GCGGCCGC CTGCAG
HixR
GAATTC GCGGCCGC T TCTAGA TTATCAAAAACCTTCCAAAAGGAAAA ACTAGT A GCGGCCGC CTGCAG
HixC
GAATTC GCGGCCGC T TCTAGA TTATCAAAAACCATGGTTTTTGATAA ACTAGT A GCGGCCGC CTGCAG
Oligos to order
HixL_top
GAATTCGCGGCCGCTTCTAGATTCTTGAAAACCAAGGTTTTTGATAAACTAGTAGCGGCCGCCTGCAG
HixL_bottom
CTGCAGGCGGCCGCTACTAGTTTATCAAAAACCTTGGTTTTCAAGAATCTAGAAGCGGCCGCGAATTC
HixR_top
GAATTCGCGGCCGCTTCTAGATTATCAAAAACCTTCCAAAAGGAAAAACTAGTAGCGGCCGCCTGCAG
HixR_bottom
CTGCAGGCGGCCGCTACTAGTTTTTCCTTTTGGAAGGTTTTTGATAATCTAGAAGCGGCCGCGAATTC
HixC_top
GAATTCGCGGCCGCTTCTAGATTATCAAAAACCATGGTTTTTGATAAACTAGTAGCGGCCGCCTGCAG
HixC_bottom
CTGCAGGCGGCCGCTACTAGTTTATCAAAAACCATGGTTTTTGATAATCTAGAAGCGGCCGCGAATTC
Building
Process of Creating Synthetic Pancake: