Team:Brasil-SP/Notebook

From 2014.igem.org

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<h1>Characterization Assemblies<h1>
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<h1>Characterization Assemblies</h1>
<p> 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.</p>
<p> 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.</p>
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<p><strong>Question</strong>: How does the transcription caused by this promoter varies with the IPTG induction?</p>
<p><strong>Question</strong>: How does the transcription caused by this promoter varies with the IPTG induction?</p>
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<p><strong>Results</strong>:</p>
<p><strong>Results</strong>:</p>
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<p>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).</p>
<p>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).</p>
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Revision as of 01:39, 9 October 2014


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!

Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions

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.

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

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.
 01/07
  • Purification of lasR PCR product
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
 03/07
  • Transformation of the Biobricks sent by the Imperial College Team:
    • BBa_K316016
    • BBa_K143031
  • Miniprep, Quantification of the plasmidial DNA samples and Restriction Analysis:
    • BBa_143012
    • BBa_K081001
    • BBa_E0840
 04/07 05/07 06/07
  • Inoculum:
    • BBa_143012
    • BBa_K081001
    • BBa_E0840
07/07
  • Miniprep, Quantification and Restriction Analysis:
    • BBa_143012
    • BBa_K081001
    • BBa_E0840
  • Transformation:
    • BBa_B0015
 08/07 09/07
  • Inoculum:
    • BBa_B0015
 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)
 11/07
  • Prepare 40% glycerol solution
  • Transformation:
    • RBS + lasR (BBa_C0079)
  • Purification:
    • RBS + qteE
 12/07 13/07
14/07
  • Assembly:AIII
    • Digestion EXSP
  • Prepare X-gal
  • Prepare more LB medium (solid + liquid)
 15/07
  • Assembly:AIII
    • Quantification of digestion EXSP products
  • Replate RBS+lasR
  • Ligation of PCR qteE in pGEM T-easy vector
  • Transformation:
    • BBa_J04450
  • Restriction analysis:
    • BBa_B0015
 16/07
  • Tranformation:
    • BBa_K823003
    • RBS+qteE
  • Electrophoresis analysis:
    • BBa_B0015
 17/07
  • Glycerol Stock and Miniprep:
    • BBa_J04450
    • RBS+lasR
    • BBa_B0015
  • Digestion:
    • BBa_J04450 (EP)
    • BBa_J04450 (XS)
  • Inoculum:
    • BBa_K823003
  • Transformation
    • BBa_K143055
 18/07
  • Glycerol Stock and Miniprep:
    • BBa_J04450
    • BBa_B0015
  • Gel Purification:
    • BBa_J04450 (EP)
    • BBa_J04450 (XS)
  • Restriction analysis:
    • BBa_K823003
 19/07 20/07
21/07
  • Transformation:
    • BBa_316016
  • Inoculum:
    • BBa_K143055
    • RBS+qteE
  • Assembly KI:
    • Digestion EXSP
 22/07
  • Inoculum:
    • BBa_K316016
  • Miniprep, Restriction analysis and Digestion:
    • RBS+qteE
  • Assemblies AIII and KI:
    • Gel Purification
    • Ligation
 23/07
  • Assemblies AIII and KI:
    • Transformation
  • Prepare more competent cells
  • Prepare LB medium (solid and liquid)
  • Inoculum
    • BBa_K316016
  • Prepare RBS+lasR and RBS+qteE for sequencing
 24/07
  • Assemblies AIII and KI:
    • Inoculum
  • Miniprep:
    • BBa_K316016
 25/07
  • Assemblies AIII and KI:
    • Glycerol Stock
    • Miniprep
    • Restriction analysis
 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:
    • Digestion EXSP
 29/07
  • >Assembly AII, AV and AVI:
    • Gel Purification
    • Ligation
 30/07
  • >Assembly AII, AV and AVI:
    • Transformation
 31/07
  • Assembly AII, AV and AVI:
    • Inoculum
  • 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

August

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/08
  • Transformation:
    • BBa_K143055
  • Assembly AII, AV and AVI:
    • Glycerol Stock
    • Miniprep
    • Restriction analysis
 02/08 03/08
04/08
  • Inoculum
    • E. coli DH5-alpha
    • KI
    • Plac BBa_K143055
  • Transformation
    • BBa_P0312
  • Assembly AV:
    • Prepare more inoculum
    • Repeat Restriction analysis
 05//08
  • Prepare assemblies KI for cytometry characterization
  • Miniprep:
    • Plac BBa_K143055
  • Inoculum
    • BBa_P0312
  • Assembly AV:
    • Miniprep
    • And once again! Restriction analysis (this BBa_K316016 is tough)
 06/08 07/08
  • MIniprep and Restriction analysis:
    • BBa_P0312
 08/08 09/08 10/08
11/08
  • Prepare Plac BBa_K143055 for sequencing
  • Analyse the RBS+qteE and RBS+lasR sequencing file
 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
 13/08
  • Assemblies BII and BIII:
    • Digestion EXSP
 14/08
  • Gel Purification and Ligation in pSB1C3 for BBiobrick standard:
    • RBS+qteE
    • RBS+lasR
  • 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
 15/08 16/08
  • Assemblies BII and BIII:
    • Digestion EXSP
 17/08
  • Assemblies BII and BIII:
    • Gel Purification
18/08
  • Assemblies BII and BIII:
    • Ligation
  • Prepare LB medium (liquid and solid)
 19/08
  • Assemblies BII and BIII:
    • Transformation
 20/08
  • RBS+qteE, RBS+lasR and Assemblies BII and BIII:
    • Inoculum
 21/08
  • RBS+qteE, RBS+lasR and Assemblies BII and BIII:
    • Glycerol Stock
    • Miniprep
    • Restriction analysis
 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)
 23/08 24/08
25/08
  • Inoculum
    • BBa_J04450
    • BBa_K143055
  • PCR BBa_K143055 with higher melting temperature (check out our Lab Journal for more info)
 26/08 27/08 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:
    • Ligation
 02/09 03/09 04/09
  • Assemblies AI, CII, KIII, KIV and KVI:
    • Glycerol Stock
    • Miniprep
    • Restriction analysis
 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
 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:
    • Digestion EXSP
 11/09
  • Prepare inoculum of the mutated BBa_K316037
  • Assemblies DI, KVIII, KX and KXI:
    • Gel Purification
    • Ligation
 12/09
  • Assemblies DI, KVIII, KX and KXI:
    • Tranformation
  • 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)
 13/09
  • Assembly KX
    • Inoculum
  • Assemblies DI, KVIII and KXI:
    • Repeat Ligation
  • Prepare LB medium (solid and liquid)
 14/09
  • Assembly KX
    • Glycerol Stock
    • Miniprep
  • Assemblies DI, KVIII and KXI:
    • Transformation
15/09
  • Assembly KX
    • Restriction analysis
  • Assemblies DI, KVIII and KXI:
    • Inoculum
  • Gel Purification of the BBa_K143015
 16/09
  • Prepare 40% glycerol solution
  • Assemblies DI, KVIII and KXI:
    • Glycerol Stock
    • Miniprep
    • Restriction analysis
 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
 18/09
  • Replate BBa_K143015. We forgot the X-gal :(
  • Measurement Interlab Study - Sample preparation and characterization by Flow Cytometry and Fluorometry
 19/09 20/09 21/09
22/09
  • Inoculum of the BBa_K143015
  • Assembly KXIV:
    • Digestão EXSP
 23/09
  • Assembly KXIV:
    • Digestão EXSP
  • Miniprep and Rstriction Analysis of the BBa_K143015
 24/09
  • Assembly KXIV:
    • Gel Purification
    • Ligation
    • Transformation
 25/09
  • Assembly KXIV
    • Inoculum
 26/09
  • Assembly KXIV:
    • Glycerol Stock
    • Miniprep
    • Restriction analysis
  • Assemnlies KII, KV and BIV:
    • Digestion EXSP
 27/09 28/09
29/09
  • Assemblies KII, KV and BIV:
    • Gel Purification
    • Ligation
    • Transformation
 30/09
  • Assemblies KII, KV and BIV:
    • Inoculum

October

Monday Tuesday Wednesday Thursday Friday Saturday Sunday
01/10
  • Assemblies KII, KV and BIV:
    • Glycerol Stock
    • Miniprep
 02/10 03/10 04/10 05/10
06/10
  • Assemblies KII, KV and BIV:
    • Restriction analysis
  • Assemblies KIX, CIII and KXVI
    • Digestão EXSP
 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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