Difference between revisions of "Part:BBa K2797002"

Latest revision as of 23:22, 17 October 2018

Composite part for streptomycin resistance

This composite part confers resistance to streptomycin. The part contains the constitutive promoter BBa_J23119 from the Anderson collection, BBa_B0034 ribosome binding site, BBa_K125520 coding sequence conferring resistance to streptomycin and BBa_K2797001 terminator.

Usage and Biology

A range of antibiotic resistance genes are required for selection of transformants when inserting foreign DNA into a chassis organism. Newcastle iGEM 2018 focused on developing a new chassis organism capable of colonising plant roots. The selected organism, Pseudomonas sp. strain CT364 was found to be resistant to a range of commonly used antibiotics. Streptomycin was one of two antibiotics Pseudomonas sp. was found not to be resistant to. The team has deposited a new streptomycin resistance cassette.

Characterisation

Figure 1 E. coli strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml).

Figure 2 E. coli strain DH5α plated on LB agar containing streptomycin (50 μg/ml).

Figure 3 E. coli strain DH5α carrying the streptomycin resistance part BBa_K2797002 plated on LB agar containing chloramphenicol (25 μg/ml). The part is in the pSB1C3 backbone conferring resistance to chloramphenicol.

Figure 4 E. coli DH5α with or without the BBa_K2797002 (SmRp) part in pSB1C3 were grown in LB broth containing streptomycin at varying concentrations. Cells were grown in 96-well plate format in 200 μl volumes at 37 C over 24 hours. (n=3 replicates, error bars are standard error of the mean).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 861
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 709
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K2797002 short</partinfo>
-This composite part confers resistance to streptomycin. The part contains the constitutive BBa_J23119 promoter, the strongest promoter of the J23100 through to J23119 promoter family
+This composite part confers resistance to streptomycin. The part contains the constitutive promoter BBa_J23119 from the Anderson collection, BBa_B0034 ribosome binding site, BBa_K125520 coding sequence conferring resistance to streptomycin and BBa_K2797001 terminator.
 ===Usage and Biology===
-A range of antibiotic resistance genes are required for selection of transformants when inserting foreign DNA into a chassis organism. Newcastle iGEM 2018 focused on developing a new chassis organism capable of colonising plant roots. The selected organism, <I>Pseudomonas fluorescens</I> DSM 25356 was found to be resistant to a range of commonly used antibiotics. Streptomycin was one of two antibiotics <I>P. fluorescens</I> was found not to be resistant to. The team therefore decided to design a streptomycin resistance part that could in future be used for selection of transformed <I>P. fluorescens</I>.
+A range of antibiotic resistance genes are required for selection of transformants when inserting foreign DNA into a chassis organism. Newcastle iGEM 2018 focused on developing a new chassis organism capable of colonising plant roots. The selected organism, <i>Pseudomonas</i> sp. strain CT364 was found to be resistant to a range of commonly used antibiotics. Streptomycin was one of two antibiotics <i>Pseudomonas</i> sp. was found not to be resistant to. The team has deposited a new streptomycin resistance cassette.
-===Part Characterisation===
+===Characterisation===
 [[File:T--NEWCASTLE--STREPTOMYCIN ECOLISMR.jpeg|500px|]]
-'''Figure 1''' <I> E. coli </I> strain DH5α transformed with streptomycin resistance part BBa_K2797002 growing on LB agar containing streptomycin at a concentration of 50 μg/ml.
+'''Figure 1''' <I> E. coli </I> strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml).
 [[File:T--NEWCASTLE--STREPTOMYCIN ECOLI.jpeg|500px|]]
-'''Figure 2''' Untransformed <I> E. coli </I> strain DH5α unable to grow on LB agar containing streptomycin at a concentration of 50 μg/ml.
+'''Figure 2''' <I> E. coli </I> strain DH5α plated on LB agar containing streptomycin (50 μg/ml).
 [[File:T--NEWCASTLE--SMRCMA.jpeg|500px|]]
-'''Figure 3''' <I> E. coli </I> strain DH5α transformed with streptomycin resistance part BBa_K2797002 growing on LB agar containing chloramphenicol at a concentration of 25 μg/ml due to resistance gene in pSB1C3 vector.
+'''Figure 3''' <I> E. coli </I> strain DH5α carrying the streptomycin resistance part BBa_K2797002 plated on LB agar containing chloramphenicol (25 μg/ml). The part is in the pSB1C3 backbone conferring resistance to chloramphenicol.
-[[File:T--NEWCASTLE--Strep.jpeg|900px|]]
+[[File:T--NEWCASTLE--Strep4.jpeg|900px|]]
-'''Figure 4''' Optical density at 600nm of three replicate wells containing LB and incrementally increasing streptomycin concentrations inoculated with <I>E. coli</I> strain DH5α untransformed and transformed with part BBa_K2797002 (SmR<I>p</i>) during incubation at 37 C over 24 hours. Control wells contained untransformed <I>E. coli</I> DH5α and an antibiotic concentration of zero. Error bars show standard error mean.
+'''Figure 4''' <I>E. coli</I> DH5α with or without the BBa_K2797002 (SmR<I>p</I>) part in pSB1C3 were grown in LB broth containing streptomycin at varying concentrations. Cells were grown in 96-well plate format in 200 μl volumes at 37 C over 24 hours. (n=3 replicates, error bars are standard error of the mean).
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