Difference between revisions of "Part:BBa K341458:Design"

Latest revision as of 02:51, 28 October 2010

Chloramphenicol resistance(with Promoter) + I-sceI site+ Recombination site

Design Notes

This part involves DraIII cutting site at two ends of the unit, this cutting site is compatible with BBF RFC 61

Dra III cutting site is "cacNNNgtg", the middle three bases is random, so the stick ends after Dra III treatment can be different.

I-sceI is an endogenous restriction enzyme, its cutting site is "tagggataacagggtaat"

This part involves DraIII cutting site at two ends of the unit, this cutting site is compatible with BBF RFC 61

Thsi part is uesd as a Insertion Fragment of Donor Plasmid in the In-vivo Recombination System of iGEM Tsinghua 2010 Project.

Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:

1) one 15bp-long restriction enzyme I-scel recognition site

2) one 25bp-long random sequence

3) one fragment for insertion and recombination

4) one 25bp-long random sequence

5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)

Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.

In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.

Source

Chloramphenicol resistance gene is form plasmid "pKD3" which is generously provided by Guoqiang CHEN of Microorganism Lab in Tsinghua University

References

Thomas E. Kuhlman and Edward C. Cox.(2009)Site-specific chromosomal integration of large synthetic constructs.Nucleic Acids Research, 2009, 1–10