Assembly

From 2006.igem.org

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:

1. Some parts are backwards in the registry - terminators, for eg.
2. Parts that are not long could be synthesized backwards.
3. 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: