Team:University of Sheffield /Wet Lab

From 2008.igem.org

(Difference between revisions)
(→Plasmids and Primers)
(→Plasmids and Primers)
Line 104: Line 104:
|BIOBRICK 1 + 2
|BIOBRICK 1 + 2
CsrA with promoter
CsrA with promoter
-
+prefix
+
|Forward '''+prefix''':
-
|Forward:
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''A ACA GAA TGT AAT GCC ATG AC - 3'   
-
5' - GAA TTC GCG GCC GCT TCT AGA ACA GAA TGT AAT GCC ATG AC - 3'   
+
|TM (50mM NaCL):  66.6 °C   
|TM (50mM NaCL):  66.6 °C   
Line 113: Line 112:
|-
|-
|CsrA without
|CsrA without
-
promoter +prefix
+
promoter
-
|Forward:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGA TGC TGA TTC TGA CTC GTC GAG TTG - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''A TGC TGA TTC TGA CTC GTC GAG TTG - 3'   
   
   
|TM (50mM NaCL):  68.8 °C   
|TM (50mM NaCL):  68.8 °C   
Line 122: Line 121:
|-
|-
|
|
-
|Reverse for both 1 & 2:
+
|Reverse for both 1 & 2 '''+suffix''':
-
5' - CTG CAG CGG CCG CTA CTA GTA GTA ACT GGA CTG CTG GGA TTT TT - 3'   
+
5' - '''CTG CAG CGG CCG CTA CTA GTA''' GTA ACT GGA CTG CTG GGA TTT TT - 3'   
|TM (50mM NaCL):  69 °C   
|TM (50mM NaCL):  69 °C   
Line 131: Line 130:
|BIOBRICK 3 + 4
|BIOBRICK 3 + 4
CrsB with
CrsB with
-
promoter +prefix
+
promoter  
-
|Forward:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGG TCG ACA GGG AGT CAG ACA AC - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''G TCG ACA GGG AGT CAG ACA AC - 3'   
|TM (50mM NaCL):  69.9 °C   
|TM (50mM NaCL):  69.9 °C   
Line 140: Line 139:
|-
|-
|CsrB without
|CsrB without
-
promoter +prefix
+
promoter  
-
|Forward:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGA GGG AGT CAG ACA ACG AAG T - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''A GGG AGT CAG ACA ACG AAG T - 3'   
|TM (50mM NaCL):  69 °C   
|TM (50mM NaCL):  69 °C   
Line 149: Line 148:
|-
|-
|
|
-
|Reverse for both 3 & 4
+
|Reverse for both 3 & 4 '''+suffix''':
-
+suffix:
+
5' - '''CTG CAG CGG CCG CTA CTA GTA''' AAT AAA AAA AGG GAG CAC TG - 3'   
-
5' - CTG CAG CGG CCG CTA CTA GTA AAT AAA AAA AGG GAG CAC TG - 3'   
+
|TM (50mM NaCL):  66.5 °C   
|TM (50mM NaCL):  66.5 °C   
Line 159: Line 157:
|BIOBRICKS 5 & 6
|BIOBRICKS 5 & 6
UvrY with
UvrY with
-
promoter +prefix
+
promoter  
-
|Forward:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGA ATG ACT AAC TAT CAG TAG CGT TAT C - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''A ATG ACT AAC TAT CAG TAG CGT TAT C - 3'   
|TM (50mM NaCL):  65.5 °C   
|TM (50mM NaCL):  65.5 °C   
Line 168: Line 166:
|-
|-
|UvrY without
|UvrY without
-
promoter +prefix
+
promoter  
-
|Forward:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGT ATT CCT TTG ATC AAC GTT CTA C - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''T ATT CCT TTG ATC AAC GTT CTA C - 3'   
   
   
|TM (50mM NaCL):  65.7 °C   
|TM (50mM NaCL):  65.7 °C   
Line 177: Line 175:
|-
|-
|
|
-
|Reverse for both 5 & 6
+
|Reverse for both 5 & 6 '''+suffix'''
-
+suffix
+
5' - '''CTG CAG CGG CCG CTA CTA GTA''' TCA CTG ACT TGA TAA TGT CT - 3'   
-
5' - CTG CAG CGG CCG CTA CTA GTA TCA CTG ACT TGA TAA TGT CT - 3'   
+
|TM (50mM NaCL):  66.3 °C   
|TM (50mM NaCL):  66.3 °C   
|GC Content:  48.8%   
|GC Content:  48.8%   
-
|Molecular Weight (Calculated):  12551.2|-
+
|Molecular Weight (Calculated):  12551.2
|-
|-
|BIOBRICK 7
|BIOBRICK 7
BarA
BarA
-
|Forward +prefix:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGC GGA ACT CCA TGA CCA ACT ACA G - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''C GGA ACT CCA TGA CCA ACT ACA G - 3'   
|TM (50mM NaCL):  69.6 °C   
|TM (50mM NaCL):  69.6 °C   
Line 195: Line 192:
|-
|-
|
|
-
|Reverse +suffix:
+
|Reverse '''+suffix''':
-
5' - CTG CAG CGG CCG CTA CTA GTA TTA CCC GAG AAT TTT GCT GG - 3'   
+
5' - '''CTG CAG CGG CCG CTA CTA GTA''' TTA CCC GAG AAT TTT GCT GG - 3'   
|TM (50mM NaCL):  68.2 °C   
|TM (50mM NaCL):  68.2 °C   
Line 204: Line 201:
|BIOBRICK 8
|BIOBRICK 8
Ori101 of pKD46
Ori101 of pKD46
-
|Forward +prefix:
+
|Forward '''+prefix''':
-
5' - GAA TTC GCG GCC GCT TCT AGA GTC TCA ATT GGT CTA GGT GAT - 3'   
+
5' - '''GAA TTC GCG GCC GCT TCT AG'''A GTC TCA ATT GGT CTA GGT GAT - 3'   
   
   
|TM (50mM NaCL):  67.3 °C   
|TM (50mM NaCL):  67.3 °C   
Line 212: Line 209:
|-
|-
|
|
-
|Reverse +suffix:
+
|Reverse '''+suffix''':
-
5' - CTG CAG CGG CCG CTA CTA GTA GTT GAT GAT ACC GCT GCC TT - 3'   
+
5' - '''CTG CAG CGG CCG CTA CTA GTA''' GTT GAT GAT ACC GCT GCC TT - 3'   
|TM (50mM NaCL):  69.8 °C   
|TM (50mM NaCL):  69.8 °C   
Line 220: Line 217:
|-
|-
|BIOBRICK 9
|BIOBRICK 9
 +
CqsA - the CAI-1
 +
producer
 +
|Forward '''+prefix''':
 +
5' - '''GAA TTC GCG GCC GCT TCT AG'''A TGA ACA AAC CGC AGC TGC C - 3' 
 +
 +
|TM (50mM NaCL):  70.4 °C 
 +
|GC Content:  57.5% 
 +
|Molecular Weight (Calculated):  12251
 +
|-
|
|
 +
|Reverse '''+suffix'''
 +
5' - '''CTG CAG CGG CCG CTA CTA GTA''' ACG GAA GTA GAA GTC ACC GT - 3' 
   
   
 +
|TM (50mM NaCL):  69.3 °C 
 +
|GC Content:  56.1% 
 +
|Molecular Weight (Calculated):  12644.2
 +
|-
 +
|BIOBRICK 10
 +
CqsS - the gene
 +
we synthesised:
 +
CAI-1 receptor
 +
|Forward '''+prefix'''
 +
5' - GAA TTC GCG GCC GCT TCT AG'''A TGA TCG TTT CTA TGG ACG T - 3' 
 +
|TM (50mM NaCL):  67 °C 
 +
|GC Content:  50% 
 +
|Molecular Weight (Calculated):  12309
 +
|-
 +
|
 +
|Reverse '''+suffix'''
 +
5' - CTG CAG CGG CCG CTA CTA GTA''' TTA AAC CCA CGC CGC AAC TT - 3' 
-
   
+
|TM (50mM NaCL):  70.4 °C 
 +
|GC Content:  56.1% 
 +
|Molecular Weight (Calculated):  12475.1
 +
|-
 +
|BIOBRICK 11
 +
Red recombinase
 +
system
 +
|Forward '''+prefix''':
 +
5' - GAA TTC GCG GCC GCT TCT AG'''A TGG ATA TTA ATA CTG AAA C - 3'  
-
   
+
|TM (50mM NaCL):  63.1 °C 
 +
|GC Content:  42.5% 
 +
|Molecular Weight (Calculated):  12319
 +
|-
 +
|
 +
|Reverse '''+suffix''':
 +
5' - '''CTG CAG CGG CCG CTA CTA GTA''' TCA TCG CCA TTG CTC CCC AA - 3'  
 +
|TM (50mM NaCL):  71.2 °C 
 +
|GC Content:  58.5% 
 +
|Molecular Weight (Calculated):  12442.1
 +
|-
 +
|BIOBRICK 12
 +
FLP recombinase
 +
|Forward '''+prefix''':
 +
5' - '''GAA TTC GCG GCC GCT TCT AG'''A TGT CTA GTT ACA TGG ATT T - 3' 
 +
 +
|TM (50mM NaCL):  64.9 °C 
 +
|GC Content:  45% 
 +
|Molecular Weight (Calculated):  12308
 +
|-
 +
|
 +
|Reverse '''+suffix''':
 +
5' - '''CTG CAG CGG CCG CTA CTA GTA''' GGT ACC TTT TTA GAA AAA TT - 3' 
 +
|TM (50mM NaCL):  64.9 °C 
 +
|GC Content:  43.9% 
 +
|Molecular Weight (Calculated):  12599.2
|}
|}

Revision as of 10:04, 29 October 2008

UniShefBanner.jpg


Introduction Our project Modelling Wet Lab Our team Timetable Miscellaneous


Introduction Protocols


Wet Lab

general plan

The Diagram is a stepwise representation of a plan we wanted to carry out to achieve our goal. First of all gene coding for BarA protein should be knocked out from wild strain of an engineered bacterium (E.coli MG1655). Then GFP should be introduced into ΔbarA mutant (MGbara1) under a promoter of a gene positively regulated by BarA. Simultaneously to those steps Fusion Kinase should be created and inserted into an expression plasmid. Then both intermediates, ΔbarA mutant with GFP in its genome (MGbara2) and plasmid with Fusion Kinase, should be combined together to give rise to the biosensor (MGfusrec). Functionality of a biosensor should be analyzed by exposing it to CAI-1 autoinducers. If the design is successful the bacterium should grow.


Click here, or on the navigation bar, for the overall timetable of our wet lab sessions.

Click here, or on the sub-navigation bar, for almost all the protocols we used in our wet lab sessions.

The lab books below contain the more detailed jobs carried out by each member of the team.

Lab Books

Dimtry's Lab Book

Eva's Lab Book

Gosia's Lab Book

Hammad's Lab Book

Rosie's Lab Book

Sam's Lab Book

Plasmids and Primers

Experimental Primers
BarA homology

sequences on pKD13 Kanamycin resistant cassette

Forward:

5' - TGA TGA TTC TGA TCC TGG CAC CGA CCG TCC TTA TTG ATT CCG GGG ATC CGT CGA CC - 3'

TM (50mM NaCL): 71.8 °C GC Content: 55.4% Molecular Weight (Calculated): 17149.1
Reverse:

5' - CGT TGA CTT CGG GCG TCA CGA CGC GAG AGG AAA TAC GTG TAG GCT GGA GCT GCT TC - 3'

TM (50mM NaCL): 72.6 °C GC Content: 58.9% Molecular Weight (Calculated): 17377.2
RFP insertion

into PGA operon = PGA homolgy on RFP cassette

Forward:

5' - TAA TTA TAC TCA CCA GCA TCA GGA GAT ATT TAT TTC CAT TAC GTA ACA TAT TTA TCC TTA TTA TTA AGC TAC TAA AGC GT - 3'

TM (50mM NaCL): 65.6 °C GC Content: 28.8% Molecular Weight (Calculated): 24492
Reverse:

5' - AAC TGG CGC GGT TTT GCT GGA TTC GGT TAT GCC GAT GGA CAA TTT AGC GAA GGA AAA GGG ATG GCT TCC TCC GAA GAC GT - 3'

TM (50mM NaCL): 73.3 °C GC Content: 51.2% Molecular Weight (Calculated): 24870.1



Biobrick Primers
BIOBRICK 1 + 2

CsrA with promoter

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA ACA GAA TGT AAT GCC ATG AC - 3'

TM (50mM NaCL): 66.6 °C GC Content: 48.8% Molecular Weight (Calculated): 12618.2
CsrA without

promoter

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA TGC TGA TTC TGA CTC GTC GAG TTG - 3'

TM (50mM NaCL): 68.8 °C GC Content: 53.3% Molecular Weight (Calculated): 13850
Reverse for both 1 & 2 +suffix:

5' - CTG CAG CGG CCG CTA CTA GTA GTA ACT GGA CTG CTG GGA TTT TT - 3'

TM (50mM NaCL): 69 °C GC Content: 52.3% Molecular Weight (Calculated): 13569.8
BIOBRICK 3 + 4

CrsB with promoter

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGG TCG ACA GGG AGT CAG ACA AC - 3'

TM (50mM NaCL): 69.9 °C GC Content: 58.5% Molecular Weight (Calculated): 12660.2
CsrB without

promoter

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA GGG AGT CAG ACA ACG AAG T - 3'

TM (50mM NaCL): 69 °C GC Content: 55% Molecular Weight (Calculated): 12395.1
Reverse for both 3 & 4 +suffix:

5' - CTG CAG CGG CCG CTA CTA GTA AAT AAA AAA AGG GAG CAC TG - 3'

TM (50mM NaCL): 66.5 °C GC Content: 48.8% Molecular Weight (Calculated): 12685.3
BIOBRICKS 5 & 6

UvrY with promoter

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA ATG ACT AAC TAT CAG TAG CGT TAT C - 3'

TM (50mM NaCL): 65.5 °C GC Content: 45.7% Molecular Weight (Calculated): 14124.2
UvrY without

promoter

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGT ATT CCT TTG ATC AAC GTT CTA C - 3'

TM (50mM NaCL): 65.7 °C GC Content: 46.5% Molecular Weight (Calculated): 13110.5
Reverse for both 5 & 6 +suffix

5' - CTG CAG CGG CCG CTA CTA GTA TCA CTG ACT TGA TAA TGT CT - 3'

TM (50mM NaCL): 66.3 °C GC Content: 48.8% Molecular Weight (Calculated): 12551.2
BIOBRICK 7

BarA

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGC GGA ACT CCA TGA CCA ACT ACA G - 3'

TM (50mM NaCL): 69.6 °C GC Content: 55.8% Molecular Weight (Calculated): 13157.6
Reverse +suffix:

5' - CTG CAG CGG CCG CTA CTA GTA TTA CCC GAG AAT TTT GCT GG - 3'

TM (50mM NaCL): 68.2 °C GC Content: 53.7% Molecular Weight (Calculated): 12592.2
BIOBRICK 8

Ori101 of pKD46

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA GTC TCA ATT GGT CTA GGT GAT - 3'

TM (50mM NaCL): 67.3 °C GC Content: 50% Molecular Weight (Calculated): 12951.4
Reverse +suffix:

5' - CTG CAG CGG CCG CTA CTA GTA GTT GAT GAT ACC GCT GCC TT - 3'

TM (50mM NaCL): 69.8 °C GC Content: 56.1% Molecular Weight (Calculated): 12568.2
BIOBRICK 9

CqsA - the CAI-1 producer

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA TGA ACA AAC CGC AGC TGC C - 3'

TM (50mM NaCL): 70.4 °C GC Content: 57.5% Molecular Weight (Calculated): 12251
Reverse +suffix

5' - CTG CAG CGG CCG CTA CTA GTA ACG GAA GTA GAA GTC ACC GT - 3'

TM (50mM NaCL): 69.3 °C GC Content: 56.1% Molecular Weight (Calculated): 12644.2
BIOBRICK 10

CqsS - the gene we synthesised: CAI-1 receptor

Forward +prefix

5' - GAA TTC GCG GCC GCT TCT AGA TGA TCG TTT CTA TGG ACG T - 3'

TM (50mM NaCL): 67 °C GC Content: 50% Molecular Weight (Calculated): 12309
Reverse +suffix

5' - CTG CAG CGG CCG CTA CTA GTA TTA AAC CCA CGC CGC AAC TT - 3'

TM (50mM NaCL): 70.4 °C GC Content: 56.1% Molecular Weight (Calculated): 12475.1
BIOBRICK 11

Red recombinase system

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA TGG ATA TTA ATA CTG AAA C - 3'

TM (50mM NaCL): 63.1 °C GC Content: 42.5% Molecular Weight (Calculated): 12319
Reverse +suffix:

5' - CTG CAG CGG CCG CTA CTA GTA TCA TCG CCA TTG CTC CCC AA - 3'

TM (50mM NaCL): 71.2 °C GC Content: 58.5% Molecular Weight (Calculated): 12442.1
BIOBRICK 12

FLP recombinase

Forward +prefix:

5' - GAA TTC GCG GCC GCT TCT AGA TGT CTA GTT ACA TGG ATT T - 3'

TM (50mM NaCL): 64.9 °C GC Content: 45% Molecular Weight (Calculated): 12308
Reverse +suffix:

5' - CTG CAG CGG CCG CTA CTA GTA GGT ACC TTT TTA GAA AAA TT - 3'

TM (50mM NaCL): 64.9 °C GC Content: 43.9% Molecular Weight (Calculated): 12599.2

Plasmids:

  • pKD46
  • pKD13
  • pCP20
  • pCQSA
  • pCQSS
Retrieved from "http://2008.igem.org/Team:University_of_Sheffield_/Wet_Lab"