Difference between revisions of "Part:BBa K1025008"

Latest revision as of 16:49, 25 September 2013

Sweet Pressure Part （RBS34）

Sweet Pressure part（RBS34） is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for providing of selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype. It is almost the same with Sweet Pressure part（RBS38）（BBa_K1025009） except for the second RBS.

Usage and Biology

This selection pressure was based on the tryptophan dependent maltose hydrolase expression which functioned in a maltose-sole carbon source culture condition. It is achieved by cloning E.coli maltose hydrolase gene (malQ) downstream of our previously constructed tryptophan biosensor（BBa_K1025005） which is controlled by the strict araBAD promoter in one malQ single deletion strain E.coli JW3379.

Functional Parameters

First of all, we tested the growth rate of JW3379 in M9 minimal culture medium with glucose and maltose as single carbon source, respectively. The results showed that with the same initial condition (the seed bacterium were from a LB culture and the initial OD600 was both set to be 0.16) after culturing in 5mL medium for about 12h in a 15mL centrifuge tube, glucose M9 medium gave a final biomass concentration of 0.58 (OD600) compared with 0.20 in maltose single carbon source M9 medium. Although this result showed that we have space to obtain tryptophan dependent growth rate by finely tuning the culture condition, due to the leakage effect of our novel tryptophan biosensor, the first rounds of trial failed. Thus, we constructed a random ribosome biding sequence (RBS) library upstream of malQ and utilized strictly controlled araBAD promoter upstream of malQ in pTRc99A vector. We applied this random library to select for the biggest growth difference between maltose and glucose M9 minimal culture condition. After the first round of selection, four strains were picked out. We further tested its performance by measuring the growth rate in different concentration of tryptophan addition. The result is shown below. It should be emphasized that even this condition is achieved by some kinds of condition test.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 2708
Illegal XbaI site found at 2735
Illegal PstI site found at 2683
Illegal PstI site found at 2747
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 2708
Illegal PstI site found at 2683
Illegal PstI site found at 2747
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 2708
Illegal BglII site found at 2105
Illegal BamHI site found at 236
Illegal BamHI site found at 2729
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 2708
Illegal XbaI site found at 2735
Illegal PstI site found at 2683
Illegal PstI site found at 2747
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 2708
Illegal XbaI site found at 2735
Illegal PstI site found at 2683
Illegal PstI site found at 2747
Illegal AgeI site found at 71
Illegal AgeI site found at 2120
Illegal AgeI site found at 2552
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2931
Illegal SapI site found at 53

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1025008 short</partinfo>
-iGEM sweet pressure part（RBS34） is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for providing of selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype.  It is almost the same with iGEM sweet pressure part（RBS38）（[https://parts.igem.org/Part:BBa_K1025009 BBa_K1025009]） except for the second RBS.
+Sweet Pressure part（RBS34） is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for providing of selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype.  It is almost the same with Sweet Pressure part（RBS38）（[https://parts.igem.org/Part:BBa_K1025009 BBa_K1025009]） except for the second RBS.
 ===Usage and Biology===
@@ Line 10: / Line 10: @@
 <partinfo>BBa_K1025008 parameters</partinfo>
 <!-- -->
-First of all, we tested the growth rate of JW3379 in M9 minimal culture medium with glucose and maltose as single carbon source, respectively. The results showed that with the same initial condition (the seed bacterium were from a LB culture and the initial OD600 was both set to be 0.16) after culturing in 5mL medium for about 12h in a 15mL centrifuge tube, glucose M9 medium gave a final biomass concentration of 0.58 (OD600) compared with 0.20 in maltose single carbon source M9 medium. Although this result showed that we have space to obtain tryptophan dependent growth rate by finely tuning the culture condition, due to the leakage effect of our novel tryptophan biosensor, the first rounds of trial failed. Thus, we constructed a random ribosome biding sequence (RBS) library upstream of malQ and utilized strictly controlled araBAD promoter upstream of malQ in pTRc99A vector. We applied this random library to select for the biggest growth difference between maltose and glucose M9 minimal culture condition. After the first round of selection, four strains were picked out. We further tested its performance by measuring the growth rate in different concentration of tryptophan addition. The result is shown below. It should be emphasized that even this condition is achieved by some kinds of condition test.
+First of all, we tested the growth rate of JW3379 in M9 minimal culture medium with glucose and maltose as single carbon source, respectively. The results showed that with the same initial condition (the seed bacterium were from a LB culture and the initial OD600 was both set to be 0.16) after culturing in 5mL medium for about 12h in a 15mL centrifuge tube, glucose M9 medium gave a final biomass concentration of 0.58 (OD600) compared with 0.20 in maltose single carbon source M9 medium. Although this result showed that we have space to obtain tryptophan dependent growth rate by finely tuning the culture condition, due to the leakage effect of our novel tryptophan biosensor, the first rounds of trial failed. Thus, we constructed a random ribosome biding sequence (RBS) library upstream of ''mal''Q and utilized strictly controlled araBAD promoter upstream of ''mal''Q in pTRc99A vector. We applied this random library to select for the biggest growth difference between maltose and glucose M9 minimal culture condition. After the first round of selection, four strains were picked out. We further tested its performance by measuring the growth rate in different concentration of tryptophan addition. The result is shown below. It should be emphasized that even this condition is achieved by some kinds of condition test.
 [[File:sweet-34-1.jpg|385px|thumb|left]]
 [[File:sweet-34-2.jpg|375px|thumb|left]]