Difference between revisions of "Part:BBa K1077000"

Revision as of 22:34, 28 September 2013

fim switch inverted repeat left IRL natural

fim switch inverted repeat left IRL natural. Ligate a part downstream, then ligate the IRR downstream of that. This switch half is in the "ON" orientation. Fig. 1 - The fim transcriptor is capable of changing states completely and unidirectionally

NEB 10-beta E. coli, which lack the native fim switch (fimS) and known fim recombinases, were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation). The state of the switch was assayed by using an asymmetric digest assay. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. The digest assay was quantified using densitometry in A and B showing greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation and fime’s previously observed functionality of catalyzing the inversion of fimS from the ON to OFF orientation. C and D show the digest assay fragments. The faint band in D, seemingly indicating residual OFF state, may correspond to the constitutive recombinase generator which is about the same size. We did not have time to cure the bacteria of the plasmid. Additionally, the switch is on the high copy pSB1C3 plasmid and so it could be that some switch plasmids are escaping recombination. We did not have time to move the switch to a low copy plasmid or the chromosome.

Sources:

1. Schwan WR. Regulation of fim genes in uropathogenic Escherichia coli. World J Clin Infect Dis 2011; 1(1): 1725.

2. I. C. Blomﬁeld, D. H. Kulasekara and B. I. Eisenstein. Integration host factor stimulates both FimB- and FimE-mediated site-speciﬁc DNA inversion that controls phase variation of type 1 ﬁmbriae expression in Escherichia coli. Molecular Microbiology (1997) 23(4), 705–717.

3. M. P. McCusker, E. C. Turner and C. J. Dorman. DNA sequence heterogeneity in Fim tyrosine-integrase recombinase-binding elements and functional motif asymmetries determine the directionality of the ﬁm genetic switch in Escherichia coli K-12. Molecular Microbiology, 67, 171–187.

4. Rice PA, Yang S, Mizuuchi K, Nash HA. Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell. 1996 Dec 27;87(7):1295-306.

5. Wang Q, Calvo JM. Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure. EMBO J. 1993 Jun;12(6):2495-501.

6. Jerome Bonnet, Pakpoom Subsoontorn, and Drew Endy. Rewritable digital data storage in live cells via engineered control of recombination directionality. PNAS. 2012 Apr 6.

7. D. L. Gally, J. Leathart and I. C. Blomﬁeld. Interaction of FimB and FimE with the ﬁm switch that controls the phase variation of type 1 ﬁmbriae in Escherichia coli K-12. Molecular Microbiology (1996) 21(4), 725–738.

8. Ham et al. A Tightly Regulated Inducible Expression System Utilizing the ﬁm Inversion Recombination Switch. Biotechnology and Bioengineering, Vol. 94, No. 1, May 5, 2006.

Sequence and Features

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]

@@ Line 3: / Line 3: @@
 fim switch inverted repeat left IRL natural. Ligate a part downstream, then ligate the IRR downstream of that. This switch half is in the "ON" orientation.
+[[File:michigemfig1.jpg]]
+Fig. 1 - The fim transcriptor is capable of changing states completely and unidirectionally
-An engineered fim switch is capable of flipping and producing protein
+NEB 10-beta E. coli, which lack the native fim switch (fimS) and known fim recombinases, were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation). The state of the switch was assayed by using an asymmetric digest assay. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. The digest assay was quantified using densitometry in A and B showing greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE).
+This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation and fime’s previously observed functionality of catalyzing the inversion of fimS from the ON to OFF orientation. C and D show the digest assay fragments. The faint band in D, seemingly indicating residual OFF state, may correspond to the constitutive recombinase generator which is about the same size. We did not have time to cure the bacteria of the plasmid. Additionally, the switch is on the high copy pSB1C3 plasmid and so it could be that some switch plasmids are escaping recombination. We did not have time to move the switch to a low copy plasmid or the chromosome.
-fig 1.
-NEB 10-beta E. coli, which lack the native fim switch (fimS) and know fim recombinases, were co-transformed with K1077003 and K1077002. When induced with aTc and grown for overnight on the left, no GFP is produced. When induced with HSL and grown overnight on the right, GFP is produced.
-The fim transcriptor is capable of changing states completely and unidirectionally
-fig. 2
-NEB 10-beta E. coli, which lack the native fim switch (fimS), were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation). E. coli expressing hbiF recombnase constitutively displayed a deep blue phenotype (A). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation. This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation.
-The state of the switch was assayed by using an asymmetric digest assay. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. The digest assay was quantified using densitometry, showing greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE).
-An inducible recombinase generator and engineered fimS make a fully functional fim transcriptor
-NEB 10-beta E. coli, which lack the native fim switch (fimS) and know fim recombinases, were co-transformed with K1077007 and K1077002
-Stock HSL was 100uM
-Stock aTc was 4.32mM
-From left to right: uninduced, .5uL stock HSL, 5uL HSL, 15uL HSL, 35uL HSL, 60uL HSL, 100uL HSL, 70uL stock aTc, 90uL stock aTc.
-Uninduced (referred to as A), 35uL HSL (referred to as B), and 90uL aTc (referred to as C) samples were washed 3 times with PBS and reinoculated without inducer, with 35uL stock HSL, and 90uL stock aTc; shown below:
-Left to right: A,B,C samples reinoculated with 35uL stock HSL; A,B,C samples reinoculated with aTc, A,B,C samples reinoculated no inducer
-Conclusions:
-We engineered the fim switch by replacing the native promoter with another promoter, namely J23100, and showed that it can flip completely in both directions and function as expected. Additionally, we submitted the switch halves separately so that other teams can flip whatever part they desire, resulting in a complete fim transcriptor system. We improved upon several natural and engineered biobrick parts. We demonstrated that the fim switch can function as a reliable and efficient biological transistor, or “transcriptor”.  Future engineers can utilize this switch for tight control of output or to store a state within DNA itself. The prospect of combining this transcriptor with the ones previously described is tantalizing in that it should enable a larger rewritable DNA storage than is currently possible. Additionally, having more switches that respond to independent sets of recombinases allows for more complex circuits to be engineered.
 Sources: