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.

A good way to make a backwards part is to PCR amplify the part from a plasmid with primers that include a XbaI site on the 3' end of the part and a SpeI on the 5' end (and four extra bases). Then the PCR product can be digested with XbaI and SpeI and directionally cloned into a vector.

For the reverse orientation of the Ara-BAD promoter (BBa_I13453), we designed these primers:

pARA-Spe-F

5’ GCAT ACTAGT ACATTGATTATTTGCACGGC 3’

pARA-Xba-R

5’ GCAT TCTAGA GCTAGCCCAAAAAAACGGTA 3’

Alternative plan for making pARA reverse uses these oligos:

pBAD_rev_Top

5’ GCAT ACTAGT ACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCC 3’

pBAD_rev_Bottom

5’ GCAT TCTAGA GCTAGCCCAAAAAAACGGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTA 3’

For the reverse orientation of the RBS - BBa_B0030 Plate DNA-1, Spot 3G, pSB1A2, we designed these primers:

RBS-back-Spe-F

5’ GCAT ACTAGT ATTAAAGAGGAGAAA 3’

RBS-back-R

5’ GCAT TCTAGA TTTCTCCTCTTTAAT 3’

For the reverse orientation of the double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I, pSB1AK3, we designed these primers:

TT-back-Spe-F

5’ GCAT ACTAGT CCAGGCATCAAATAAAACGA

TT-back-R

5’ GCAT TCTAGA TATAAACGCAGAAAGGCCCA

Several other 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.
4. Can cut out a part with NotI, then religate in both orientations.

Examples

BBa_B0022 Terminator (Reverse B0012) Length: 84 Base Pairs Including BioBrick Ends

BBa_P1004 is ampR resistance cassette in reverse orientation Length: 984 Base Pairs Including BioBrick Ends (In planning stages only)

Bba_B0034 is a RBS with the sequence:

gaattcgcggccgcttctagag attaaagaggagaaa tactagtagcggccgctgcag

It could be redesigned with the RBS reversed:

gaattcgcggccgcttctagag tttctcctctttaat tactagtagcggccgctgcag

Oligos for reverse RBS that produce EcoRI and SpeI sticky ends:

5’ AATTC GCGGCCGC T TCTAGA G TTTCTCCTCTTTAAT T A 3’

5’ CTAGT A ATTAAAGAGGAGAAA C TCTAGA A GCGGCCGC G 3’

Here is a proposed strategy for making backwards parts flanked by hix sites:

hix BioBricks

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 have looked at so far have 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

AATTCGCGGCCGCTTCTAGATTCTTGAAAACCAAGGTTTTTGATAAACTAGTAGCGGCCGCCTGCA

HixL_bottom

GGCGGCCGCTACTAGTTTATCAAAAACCTTGGTTTTCAAGAATCTAGAAGCGGCCGCG

HixR_top

AATTCGCGGCCGCTTCTAGATTATCAAAAACCTTCCAAAAGGAAAAACTAGTAGCGGCCGCCTGCA

HixR_bottom

GGCGGCCGCTACTAGTTTTTCCTTTTGGAAGGTTTTTGATAATCTAGAAGCGGCCGCG

HixC_top

AATTCGCGGCCGCTTCTAGATTATCAAAAACCATGGTTTTTGATAAACTAGTAGCGGCCGCCTGCA

HixC_bottom

GGCGGCCGCTACTAGTTTATCAAAAACCATGGTTTTTGATAATCTAGAAGCGGCCGCG

Building

Biobricking in one hix site:

Example of a construct for one flip (terminator, although CDS would be better):

Biobricking hix sites onto parts:

Assembly of One Pancake Test Construct:

Assembly of Two Pancake Test Construct would be next. Probably want two genes.

Assembly of Multiple Pancake Test Constructs:

First Transformations:

>1. Terminator T1 Bba_B0010 DNA-2 3P<

2. Promoter Pbad BBa_I13453

>3. Promoter w/RBS (TetR Repressed) BBa_J13002 DNA-2 3F <

4. Reverse Amp^r BBa_P1004 (Oops, planning)

5. Reverse CmR BBa_P1009 789 bp (Oops, planning)

>6. Double Forward Terminator BBa_B0015 DNA-1 1I <

>7. Tet^r Bba_P1001 DNA-2 23F <

Questions:

Will the TetR repressed promoter in BBa_J13002 be always on in JM109 cells?

Do we want to be able to turn on a Start Promoter(i.e. LacP using IPTG) on/off so recombination occurs 1st; and then use a different inducible promoter to turn on transcription?