Evry-Saclay 2021 iGEM team website

Parts

Collection

Parts Collection

In our project we developed the Evolution.T7 targeted evolution tool, which uses the processivity of T7 RNA polymerase which specifically recognises a promoter orthogonal to the E. coli transcription machinery, goes over a DNA sequence of interest during a transcription step and stops at a specific terminator. By linking this T7RNAP to a base modifying domain, mutations are introduced randomly along the sequence as the T7RNAP passes over it.

Our part collection includes fusions of the wild-type T7RNAP with a collection of deaminase proteins as mutators, but also of a mutant T7RNAP-CGG-R12-KIRV [1] that transcribes from the mutant T7_CGG promoter. Our collection allows modularity considering the possibility to mix and match different mutators to achieve different types of mutations at different rates.

Moreover, using the Evolution.T7 system, we generated a second collection comprising more than thirty variants of the ampicillin-resistance gene AmpR able to confer resistance to aztreonam, a different antibiotic

Mutators in Forward Orientation

	Mutator's Part Number	Mutator's expression cassettes Part Number
T7RNAP	-	GenBank MN450171 [2]
AID T7RNAP	-	GenBank MN450172 [2]
pmCDA1 T7RNAP	-	GenBank MN450173 [2]
rAPOBEC1 T7RNAP	-	GenBank MN450174 [2]
TadA* T7RNAP	-	GenBank MN450175 [2]
evoAPOBEC1-BE4max T7RNAP	BBa_K3766008	BBa_K3766108
evoCDA1-BE4max T7RNAP	BBa_K3766009	BBa_K3766109
ABE8.20-m T7RNAP	BBa_K3766010	BBa_K3766110

Mutators in Reverse Orientation

	Mutator's Part Number	Mutator's expression cassettes Part Number
T7RNAP CGG-R12-KIRV	BBa_K3766000	BBa_K3766100
AID T7RNAP CGG-R12-KIRV	BBa_K3766001	BBa_K3766101
pmCDA1 T7RNAP CGG-R12-KIRV	BBa_K3766002	BBa_K3766102
rAPOBEC1 T7RNAP CGG-R12-KIRV	BBa_K3766003	BBa_K3766103
TadA* T7RNAP CGG-R12-KIRV	BBa_K3766004	BBa_K3766104
evoAPOBEC1-BE4max T7RNAP CGG-R12-KIRV	BBa_K3766005	BBa_K3766105
evoCDA1-BE4max T7RNAP CGG-R12-KIRV	BBa_K3766006	BBa_K3766106
ABE8.20-m T7RNAP CGG-R12-KIRV	BBa_K3766007	BBa_K3766107

The collection of AmpR variants evolved to confer resistance to aztreonam

Aztreonam resistance gene	Part Number	Expression cassettes Part Number
AZT-1	BBa_K3766051	BBa_K3766151
AZT-2	BBa_K3766052	BBa_K3766152
AZT-3	BBa_K3766053	BBa_K3766153
AZT-4	BBa_K3766054	BBa_K3766154
AZT-5	BBa_K3766055	BBa_K3766155
AZT-6	BBa_K3766056	BBa_K3766156
AZT-7	BBa_K3766057	BBa_K3766157
AZT-8	BBa_K3766058	BBa_K3766158
AZT-9	BBa_K3766059	BBa_K3766159
AZT-10	BBa_K3766060	BBa_K3766160
AZT-11	BBa_K3766061	BBa_K3766161
AZT-12	BBa_K3766062	BBa_K3766162
AZT-13	BBa_K3766063	BBa_K3766163
AZT-14	BBa_K3766064	BBa_K3766164
AZT-15	BBa_K3766065	BBa_K3766165
AZT-16	BBa_K3766066	BBa_K3766166
AZT-17	BBa_K3766067	BBa_K3766167
AZT-18	BBa_K3766068	BBa_K3766168
AZT-19	BBa_K3766069	BBa_K3766169
AZT-20	BBa_K3766070	BBa_K3766170
AZT-21	BBa_K3766071	BBa_K3766171
AZT-22	BBa_K3766072	BBa_K3766172
AZT-23	BBa_K3766073	BBa_K3766173
AZT-24	BBa_K3766074	BBa_K3766174
AZT-25	BBa_K3766075	BBa_K3766175
AZT-26	BBa_K3766076	BBa_K3766176
AZT-27	BBa_K3766077	BBa_K3766177
AZT-28	BBa_K3766078	BBa_K3766178
AZT-29	BBa_K3766079	BBa_K3766179
AZT-30	BBa_K3766080	BBa_K3766180
AZT-31	BBa_K3766081	BBa_K3766181
AZT-32	BBa_K3766082	BBa_K3766182
AZT-33	BBa_K3766083	BBa_K3766183
AZT-34	BBa_K3766084	BBa_K3766184
AZT-35	BBa_K3766085	BBa_K3766185

References

[1] Meyer AJ, Ellefson JW, Ellington AD. Directed evolution of a panel of orthogonal T7 RNA polymerase variants for in vivo or in vitro synthetic circuitry. ACS synthetic biology (2015) 4: 1070–1076.

[2] Álvarez B, Mencía M, de Lorenzo V, Fernández LÁ. In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9. Nature Communications (2020) 11: 6436.