From 2014.igem.org
WELCOME TO iGEM 2014!
Your team has been approved and you are ready to start the iGEM season!
On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world!
Click here to edit this page!
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Notebook |
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
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Main Assembly Map
Assemblies forms
Assembly form template
This is the template designed to help with the laboratory organization during the assemble of biological parts. If you want to use the assembly method we used you can print the form and complete it for each construction. Hope it helps :)
Characterization Assemblies
Apart from the main genetic circuit we also assembled others for characterization purposes, such as the validation of the promoters and tunning of our threshold setter concentration, the QteE.
Promoter BBa_K823003
Question: Does the constitutive promoter BBa_K823003 work properly?
Results: After the incubation period of the transformed E. coli a large portion of the colonies were glowing green. So the promoter does work. Moreover, this biobrick works on E. coli despite the fact it was designed for B. subtilis.
Promoter BBa_K143015
Question: How does the transcription caused by this promoter varies with the IPTG induction?
Results:
Tunning of the QteE Threshold
Question:What are the concentration of QteE needed to hamper the LasR induction of the promoter PlasR?
This is the most difficult task of our project. Tunning the production of QteE so that we establish the correct threshold for the discretization of the Cystatin C level in serum. To attack this challenge we designed 3 circuits so that we could plot a calibration curve. In this circuits we put the transcription of the LasR and QteE under two differnt promoters, the Pveg (BBa_K823003) and PlasR (BBa_K143015).
Assembly Forms
Life Inside the LAB
July
Monday |
Tuesday |
Wednesday |
Thursday |
Friday |
Saturday |
Sunday |
30/06
- PCR lasR (BBa_C0079). The purpose of this PCR was to add the RBS (BBa_K143021), through addition of the sequence in the primer foward, and also to remove the LVA tag. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion.
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01/07
- Purification of lasR PCR product
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02/07
- Transformation of the Biobricks:
- BBa_143012
- BBa_K081001
- BBa_E0840
- Inoculum of the Biobricks sent by iGEM HQ:
- BBa_K316037
- BBa_K316018
- BBa_K316015
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03/07
- Transformation of the Biobricks sent by the Imperial College Team:
- Miniprep, Quantification of the plasmidial DNA samples and Restriction Analysis:
- BBa_143012
- BBa_K081001
- BBa_E0840
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04/07 |
05/07 |
06/07
- Inoculum:
- BBa_143012
- BBa_K081001
- BBa_E0840
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07/07
- Miniprep, Quantification and Restriction Analysis:
- BBa_143012
- BBa_K081001
- BBa_E0840
- Transformation:
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08/07 |
09/07
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10/07
- Miniprep, Quantification and Restriction Analysis:
- BBa_B0015 (restriction analysis fail, repeat)
- PCR the qteE gene for amplification. We also added the RBS (BBa_K143021) using the foward primer. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion.
- Ligation of RBS+lasR in PTZ57R/T vector (instaclone kit)
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11/07
- Prepare 40% glycerol solution
- Transformation:
- Purification:
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12/07 |
13/07 |
14/07
- Assembly:AIII
- Prepare X-gal
- Prepare more LB medium (solid + liquid)
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15/07
- Assembly:AIII
- Quantification of digestion EXSP products
- Replate RBS+lasR
- Ligation of PCR qteE in pGEM T-easy vector
- Transformation:
- Restriction analysis:
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16/07
- Tranformation:
- Electrophoresis analysis:
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17/07
- Glycerol Stock and Miniprep:
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- BBa_J04450
- RBS+lasR
- BBa_B0015
- Digestion:
- BBa_J04450 (EP)
- BBa_J04450 (XS)
- Inoculum:
- Transformation
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18/07
- Glycerol Stock and Miniprep:
- Gel Purification:
- BBa_J04450 (EP)
- BBa_J04450 (XS)
- Restriction analysis:
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19/07 |
20/07 |
21/07
- Transformation:
- Inoculum:
- Assembly KI:
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22/07
- Inoculum:
- Miniprep, Restriction analysis and Digestion:
- Assemblies AIII and KI:
- Gel Purification
- Ligation
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23/07
- Assemblies AIII and KI:
- Prepare more competent cells
- Prepare LB medium (solid and liquid)
- Inoculum
- Prepare RBS+lasR and RBS+qteE for sequencing
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24/07
- Assemblies AIII and KI:
- Miniprep:
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25/07
- Assemblies AIII and KI:
- Glycerol Stock
- Miniprep
- Restriction analysis
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26/07 |
27/07 |
28/07
- Assembly AIII:
- Restriction analysis (+NdeI enzyme)
- OBS: This analysis was reapeted because we had problems confirming the assembly. The issue was that bot the construction and the pSB1C3 vector had similar size. Check out the Lab Journal report of the day for more information.
- Assembly AII,
AV and
AVI:
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29/07
- >Assembly AII,
AV and
AVI:
- Gel Purification
- Ligation
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30/07
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31/07
- Assembly AII,
AV and
AVI:
- PCR BBa_K143055 for RBS removal
- OBS: This was one of the parts sent by the Imperial College Team. Because of a comunication failure we assumed that this part was comming without the RBS, so we included a RBS in the parts we were going to use with this Lac promotor :)
- PCR of Plac BBa_K143055 was followed by a Gel Purification and Ligation in the PUC19 vector
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August
Monday |
Tuesday |
Wednesday |
Thursday |
Friday |
Saturday |
Sunday |
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01/08
- Transformation:
- Assembly AII,
AV and
AVI:
- Glycerol Stock
- Miniprep
- Restriction analysis
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02/08 |
03/08 |
04/08
- Inoculum
- E. coli DH5-alpha
- KI
- Plac BBa_K143055
- Transformation
- Assembly AV:
- Prepare more inoculum
- Repeat Restriction analysis
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05//08
- Prepare assemblies KI for cytometry characterization
- Miniprep:
- Inoculum
- Assembly AV:
- Miniprep
- And once again! Restriction analysis (this BBa_K316016 is tough)
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06/08 |
07/08
- MIniprep and Restriction analysis:
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08/08 |
09/08 |
10/08 |
11/08
- Prepare Plac BBa_K143055 for sequencing
- Analyse the RBS+qteE and RBS+lasR sequencing file
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12/08
- Digestion:
- RBS+qteE
- RBS+lasR
- OBS: this digestion was performed so we clone this parts in the pSB1C3 vector to put them in Biobrick standard
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13/08
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14/08
- Gel Purification and Ligation in pSB1C3 for BBiobrick standard:
- Assemblies BII and
BIII:
- Gel Purification
- OBS: we had some problems here with the purification and we lost all our digestions, so we're repeating the digestion
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15/08 |
16/08
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17/08
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18/08
- Assemblies BII and
BIII:
- Prepare LB medium (liquid and solid)
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19/08
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20/08
- RBS+qteE, RBS+lasR and Assemblies BII and
BIII:
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21/08
- RBS+qteE, RBS+lasR and Assemblies BII and
BIII:
- Glycerol Stock
- Miniprep
- Restriction analysis
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22/08
- Prepare LB medium (liquid and solid)
- Assembly BII:
- Repeat restriction analysis adding the enzyme NdeI (for more info check out the Lab Journal)
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23/08 |
24/08 |
25/08
- Inoculum
- PCR BBa_K143055 with higher melting temperature (check out our Lab Journal for more info)
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26/08
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27/08
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28/08 |
29/08 |
30/08 |
31/08 |
September
Monday |
Tuesday |
Wednesday |
Thursday |
Friday |
Saturday |
Sunday |
01/09
- Assembly CII
- Digestion of the part BII with EcoRV (for more info check out our Lab Journal)
- Assemblies AI,
CII,
KIII,
KIV and
KVI:
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02/09
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03/09
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04/09
- Assemblies AI,
CII,
KIII,
KIV and
KVI:
- Glycerol Stock
- Miniprep
- Restriction analysis
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05/09 |
06/09 |
07/09 |
08/09
- QuickChange PCR to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site
- Transformation of the QuickCHange PCR product
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09/09 |
10/09
- The QuickChange protocol didn't work well. So try again with more attention!
- QuickChange PCR to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site
- Transformation of the QuickCHange PCR product
- Assemblies DI,
KVIII,
KX and
KXI:
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11/09
- Prepare inoculum of the mutated BBa_K316037
- Assemblies DI,
KVIII,
KX and
KXI:
- Gel Purification
- Ligation
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12/09
- Assemblies DI,
KVIII,
KX and
KXI:
- We had far to many difficulties trying to remove the RBS from the BBa_K143055, so we decided to synthesize it using PCR.
- PCR for BBa_143015 (BBa_143055 without RBS)
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13/09
- Assembly KX
- Assemblies DI,
KVIII and
KXI:
- Prepare LB medium (solid and liquid)
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14/09
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15/09
- Assembly KX
- Assemblies DI,
KVIII and
KXI:
- Gel Purification of the BBa_K143015
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16/09
- Prepare 40% glycerol solution
- Assemblies DI,
KVIII and
KXI:
- Glycerol Stock
- Miniprep
- Restriction analysis
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17/09
- LIgation of the synthesized BBa_K143015 in TOPO vector followed by Transformation
- Measurement Interlab Study - Samples growth biobrick devices 1, 2 and 3
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18/09
- Replate BBa_K143015. We forgot the X-gal :(
- Measurement Interlab Study - Sample preparation and characterization by Flow Cytometry and Fluorometry
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19/09 |
20/09 |
21/09 |
22/09
- Inoculum of the BBa_K143015
- Assembly KXIV:
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23/09
- Assembly KXIV:
- Miniprep and Rstriction Analysis of the BBa_K143015
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24/09
- Assembly KXIV:
- Gel Purification
- Ligation
- Transformation
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25/09
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26/09
- Assembly KXIV:
- Glycerol Stock
- Miniprep
- Restriction analysis
- Assemnlies KII, KV and BIV:
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27/09 |
28/09 |
29/09
- Assemblies KII, KV and BIV:
- Gel Purification
- Ligation
- Transformation
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30/09
- Assemblies KII, KV and BIV:
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October
Monday |
Tuesday |
Wednesday |
Thursday |
Friday |
Saturday |
Sunday |
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01/10
- Assemblies KII, KV and BIV:
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02/10 |
03/10 |
04/10 |
05/10 |
06/10
- Assemblies KII, KV and BIV:
- Assemblies KIX, CIII and KXVI
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07/10 |
08/10 |
09/10 |
10/10 |
11/10 |
12/10 |
13/10 |
14/10 |
15/10 |
16/10 |
17/10 |
18/10 |
19/10 |
20/10 |
21/10 |
22/10 |
23/10 |
24/10 |
25/10 |
26/10 |
27/10 |
28/10 |
29/10 |
30/10 |
31/10 |
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