Difference between revisions of "Part:BBa J31001:Design"

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===References===
 
===References===
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[https://dspace.mit.edu/handle/1721.1/21168|  Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]
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*[https://dspace.mit.edu/handle/1721.1/21168|  Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]
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*Li, W., Kamtekar, S., Xiong, Y., Sarkis, G.J., Grindley, N.D., Steitz, T.A. (2005) ''Structure of a synaptic gamma delta resolvase tetramer covalently linked to two cleaved DNAs''. Science. v309 pp.1210-1215 , 2005

Revision as of 03:57, 29 October 2006

DNA invertase Hin tagged with LVA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part is cloned in plasmid pSB1A2.

The Biobricks on this part are not wildtype but the cut sites are still viable.

BioBrick Prefix
There is no T spacer between the NotI site and the XbaI site. There is no G spacer between the XbaI and the coding region. 		GAATTCGCGGCCGC-TCTAGA-
Hin coding TGGCTACTATTGGGTATATTCGGGTGTCAACAATTGACCAAAATATCGAT

TTACAGCGTAATGCGCTTACCAGTGCAAATTGTGACCGCATTTTTGAGGA
CCGTATCAGTGGCAAGATTGCAAACCGCCCCGGCCTGAAACGAGCGTTAA
AGTATGTAAATAAAGGCGATACTCTTGTCGTCTGGAAATTAGACAGACTG
GGCCGCAGCGTGAAAAACCTGGTGGCGTTAATATCAGAATTACATGAACG
TGGAGCTCACTTCCATTCTTTAACCGATAGTATTGATACCAGTAGCGCGA
TGGGGCGATTCTTTTTTCATGTAATGTCAGCACTGGCCGAGATGGAGCGA
GAATTAATTGTCGAGCGAACCCTTGCCGGACTGGCTGCCGCCAGAGCGCA
AGGACGACTGGGAGGGCGCCCTCGGGCGATCAACAGACATGAACAGGAAC
AGATTAGTCGGCTATTAGAGAAAGGCCATCCTCGGCAGCAACTAGCTATT
ATTTTTGGTATTGGCGTATCTACCTTATACAGATATTTTCCGGCAAGCCG
TATAAAAAAACGAATGAATAGGCCTGCTGCAAACGACGAAAACTACGCTT
TAGTAGCTTA

BioBrick Suffix: There is no T spacer between the insert and the SpeI site. The T spacer between the SpeI and the NotI sites should be an A. The last C of the NotI site is not conserved with the initial C from the PstI site. The BB suffix currently has this sequence for the NotI and PstI sites GCGGCCGcCTGCAG But it should have been: GCGGCCGCTGCAG -ACTAGTTGCGGCCGCCTGCAG

We compared our BioBricks with those from Tom Knight's paper, Idempotent Vector Design for Standard Assembly of Biobricks. As seen below

BioBricks from paper.png

Data

HinLVA has been assembled with a pLac promoter and RBS (see BBa_S03536) to create a HinLVA expression casette. We observe inversion of HixC-flanked segments of DNA in the presence of this casette. Inversion occurs with induction of Hin expression. This may be caused by read-through from the vector backbone or leaky transcription from pLac.

3-D structure of a Hin protein complex bound to DNA. View the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ZR4 interactive 3-D Jmol image].
 next image next image

Source

Salmonella typhimurium and the the Hin part without LVA.

References