Team:University of Sheffield /Wet Lab

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!align="center"|[[Team:University_of_Sheffield /Misc| Miscellaneous]]
!align="center"|[[Team:University_of_Sheffield /Misc| Miscellaneous]]
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{| style="color:#888888;background-color:##888888;" cellpadding="1" cellspacing="1" border="4" bordercolor=#888888 width="35%" align="center"
!align="center"|[[Team:University_of_Sheffield /Wet Lab|Introduction]]
!align="center"|[[Team:University_of_Sheffield /Wet Lab|Introduction]]
!align="center"|[[Team:University_of_Sheffield /Protocols|Protocols]]
!align="center"|[[Team:University_of_Sheffield /Protocols|Protocols]]
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=Wet Lab=
=Wet Lab=
-
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__TOC__
+
 
 +
 
 +
Click [[Team:University_of_Sheffield /Timetable|here]], or on the navigation bar, for the overall timetable of our wet lab sessions.
 +
 
 +
Click [[Team:University_of_Sheffield /Protocols|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.
 +
 
 +
==Overview==
 +
 
 +
[[Image:general_plan.jpg|600px|center|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.
 +
 
 +
 
==Lab Books==
==Lab Books==
<!-- Nasty HTML hack to get the gallery centred -->
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Image:Dmitry_shef.jpg|[[Team:University_of_Sheffield/Dimtry | Dimtry's Lab Book]]
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Image:Dmitry_shef.jpg|[[Team:University_of_Sheffield/Dimtry | Dmitry's Lab Book]]
Image:Eva_shef.jpg|[[Team:University_of_Sheffield/Eva | Eva's Lab Book]]
Image:Eva_shef.jpg|[[Team:University_of_Sheffield/Eva | Eva's Lab Book]]
Image:Gosia_shef.jpg|[[Team:University_of_Sheffield/Gosia | Gosia's Lab Book]]
Image:Gosia_shef.jpg|[[Team:University_of_Sheffield/Gosia | Gosia's Lab Book]]
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==Protocols==
+
==Plasmids and Primers==
-
[[Our Protocols]]
+
{| align="center" border="1"
 +
! colspan="5"|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' 
-
===Tecan Fluorescence Measurement===
+
|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:
-
Protocol used for characterization :
+
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' 
-
1. Grow 5ml of overnight cultures of DH5-alpha cells containing the plasmid with GFP-LVA.
+
|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' 
-
2. Resuspend the overnight cultures in 50 ml of LB medium until the OD600 reaches about 0.6.
+
|TM (50mM NaCL):  73.3 °C 
 +
|GC Content:  51.2% 
 +
|Molecular Weight (Calculated):  24870.1
 +
|}
 +
 
 +
 
 +
 
 +
 
 +
 
 +
{| align="center" border="1"
 +
! colspan="5"|Biobrick Primers
 +
|-
 +
|BIOBRICK 1 + 2
 +
CsrA with promoter
 +
|Forward '''+prefix''':
 +
5' - '''GAA TTC GCG GCC GCT TCT AG'''A 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 AG'''A TGC TGA TTC TGA CTC GTC GAG TTG - 3' 
   
   
-
3. Quickly add 0.2 mM of IPTG to the medium and plate that into 96 well plate.
+
|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' 
-
4.     To 96 well plate add 180 ul of LB + IPTG as a control, 180 ul of DH5-alpha cells not induced with IPTG, finally add 180 ul of DH5-alpha induced with IPTG. Carry out the aforementioned in duplicates.
+
|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 AG'''G TCG ACA GGG AGT CAG ACA AC - 3' 
-
5. Use the high flow cytometry machine Tecan to measure the fluorescence every 15 minutes for 8 hours (excitation wv – 485 nm, emission wv- 535 nm )(including shaking = aeration of the cultures 2 minutes prior each measurement)
+
|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 AG'''A GGG AGT CAG ACA ACG AAG T - 3' 
-
=Timetable=
+
|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' 
-
===Dry Lab Period ===
+
|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 AG'''A 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 AG'''T ATT CCT TTG ATC AAC GTT CTA C - 3' 
   
   
-
'''February 2008'''
+
|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' 
-
The very beginning. Three team members, Gosia, Eva and Dmitry met each other to discuss the possibility of joining iGEM competition this year. Straight after that Rosie joined the team.  
+
|TM (50mM NaCL):  66.3 °C 
-
March 2008 – The idea to participate in iGEM was presented in the Department of Molecular Biology and Biotechnology (MBB)  in the students and staff committee meeting. The head of MBB Department, Prof. D. Hornby agreed to help us in managing this project.
+
|GC Content:  48.8% 
 +
|Molecular Weight (Calculated): 12551.2
 +
|-
 +
|BIOBRICK 7
 +
BarA
 +
|Forward '''+prefix''':
 +
5' - '''GAA TTC GCG GCC GCT TCT AG'''C GGA ACT CCA TGA CCA ACT ACA G - 3' 
-
'''April 2008'''
+
|TM (50mM NaCL):  69.6 °C 
-
*The team now consisted of 9 members, mostly joined by MBB Level 2 students. The early brainstorming took place. However, no ideas that would be feasible to complete during the summer and would have actual impact on science appeared.  
+
|GC Content: 55.8% 
-
*A new science orientated society was established within the University of Sheffield Union of Students. The initial purpose of the SynBio society was to help students within the University to participate in iGEM next year, along with that a Scientific Journal Club with monthly meetings is on the agenda
+
|Molecular Weight (Calculated):  13157.6
-
* Applications for Funding to various sources were sent out.
+
|-
-
'''June 2008'''
+
|
-
* the idea of sensing biological water contamination employing the Quorum Sensing mechanism was finalised
+
|Reverse '''+suffix''':
-
* 15th June - meeting with Prof. P. Wright took place to discuss a possibility to join labs in the Bioincubator, a new business and research facility in Sheffield
+
5' - '''CTG CAG CGG CCG CTA CTA GTA''' TTA CCC GAG AAT TTT GCT GG - 3' 
-
* 29th June - two level 2 students representing the Department of Automatic Control and Systems Engineering joined the team to cover the Engineering parts of the project.
+
-
* 30th June - meeting with Dr. Catherine Biggs from the ChELSI group took place to discuss a possible approach towards using the Quorum sensing as a way of sensing water contamination
+
-
'''July 2008'''
+
-
* 2nd to 16th July - team members representing biological and engineering sides of the project introduced each other to their plans of action as well as explained the material behind it.
+
-
* 7th July - meeting with Prof. Marian Gheorghe from Automatic Control and Systems Engineering. The meeting discussed the approach towards mathematical modeling of the Quorum Sensing using E.coli as a tool.
+
-
* iCHEME offered a financial contribution towards the trip to Jamboree.
+
-
* Discussions with the group as well as Dr. C. Biggs, with an aim to choose the right model organism to represent water pathogens.
+
-
* Ph.D. student Esther Karunakaran proposed using a fusion histidine kinase as a receptor for V. cholerae  autoinducer . V. cholerae , one of the causative agents of water borne diseases, was then chosen as a model organism representing water pathogens.
+
-
* The native E.coli BarA - UvrY signal transduction pathway was selected for "hijacking" by our fusion kinase.
+
-
* 20th July -  students discussed a possible target for GFP insertion in the E.coli genome, the pgaABCD operon was chosen.
+
-
* Prof. P. Wright and Dr. C. Biggs representing the ChELSI research group within the University of Sheffield approved the project plan and agreed to provide lab materials and lab space in the Bioincubator research facility.
+
-
* IDT DNA kindly agreed on a contribution of £1000 towards the cost of gene synthesis and primer orders. The CqsS – a native receptor for CAI-1 molecules was ordered for further experiments.
+
-
'''August 2008'''
+
-
* 4th August - meeting with David Wengraf to discuss possible biohazard before final approval to enter Bioincuabotr Labs.
+
-
* 4th and 5th August - meetings with Prof. Rice so as to choose a correct place to cut the CqsS and BarA to have a functional fusion kinase.
+
-
=== Wet Lab Period ===
+
|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 AG'''A GTC TCA ATT GGT CTA GGT GAT - 3' 
   
   
-
'''Mid - August 2008'''
+
|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' 
-
* 7th August - team entered the labs to start actual experiments. Preparation for gene knockout using Datsenko and Wanner method was the first on the agenda.  Prof. Stafford from the Univeristy of Sheffield Medical School provided us with electroporation protocol. From 11th August to mid September the electroporation was carried out unsuccessfully.
+
|TM (50mM NaCL):  69.8 °C 
-
* 15th August - an official project presentation to the ChELSI research group.
+
|GC Content:  56.1%  
-
* the transformation using the Biobrick BBa_I763004  containing a plasmid with GFP – LVA tag  was made. All attempts were unsuccessful despite using pre – approved protocols and cultures.
+
|Molecular Weight (Calculated):  12568.2
-
* 26th August - Mr. Peter Grant representing the Biofusion Group provided funding to cover the costs of the U. S. VISA for 3 team members.
+
|-
 +
|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' 
-
'''September 2008'''  
+
|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' 
-
* The gene order for CqsS membrane receptor arrived.
+
|TM (50mM NaCL):  67 °C 
-
* Along with that Prof. B. Bassler from Princeton University sent us the plasmid containing CqsA - a protein producing CAI -1 (Cholera Autoinducer – 1) with the appropriate purification protocol.
+
|GC Content:  50% 
-
* Prof. O. Melefor from Karolinska Instutue kindly provided us with a DH5 – alpha strain containing a barA knockout
+
|Molecular Weight (Calculated):  12309
-
* Prof. Robert Poole agreed to cover further funding for the trip to USA. On 16th September the hotels and flights to the Jamboree were booked, along with registration confirmation of all attending team members.   
+
|-
-
* The electroporation and transformation were still unsuccessful even after extensive troubleshooting.  
+
|
-
* 29th September VISAs were attained.  
+
|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
 +
|}
-
'''October 2008'''
+
Plasmids:
-
* Plasmid prep of one possible GFP - LVA transformant colony was made. The attempt was to characterize a possible Biobrick using Tecan as well as loading a restriction digest of the plasmid prep on the agarose gel. The results suggested that the colony was most likely contamination.
+
[[Image:pKD46.png]]
-
* 8th to 15th October -preparation for Biobrick submission to the Parts Registry took place. Failed transformations raise suspicions of a faulty Biobrick booklet. Thorough investigation of various DNA parts from all over the booklet proves this hypothesis. Due to faulty DNA shipment no parts can be submitted to the Registry as the deadline for variants was 8th October.
+
[[Image:pKD13.png]]
 +
*pCP20 - "an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis" (Datsenko and Wanner,[4])
 +
*pCQSA - plasmid sent to us by Bonnie Bassler's lab, contains the CAI-1 producing gene
 +
*pCQSS - plasmid synthesised by us (via idtDNA) containing the gene for the CAI-1 receptor

Latest revision as of 01:49, 30 October 2008

UniShefBanner.jpg


Introduction Our project Modelling Wet Lab Our team Timetable Miscellaneous


Introduction Protocols


Contents

Wet Lab

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.

Overview

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.


Lab Books

Dmitry'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.png PKD13.png

  • pCP20 - "an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis" (Datsenko and Wanner,[4])
  • pCQSA - plasmid sent to us by Bonnie Bassler's lab, contains the CAI-1 producing gene
  • pCQSS - plasmid synthesised by us (via idtDNA) containing the gene for the CAI-1 receptor
Retrieved from "http://2008.igem.org/Team:University_of_Sheffield_/Wet_Lab"