Team:Brasil-SP/Notebook

From 2014.igem.org

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<h1>Assembly Map</h1>
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<h1>Main Assembly Map</h1>
<!--estou colocando os fichamentos dos assemblies aqui, ainda preciso acabar os que faltam mas ja da pra experimentar com a organização destes-->
<!--estou colocando os fichamentos dos assemblies aqui, ainda preciso acabar os que faltam mas ja da pra experimentar com a organização destes-->
<img src="https://static.igem.org/mediawiki/2014/8/87/Mapa.png" style="width:100%;height:900px"/>
<img src="https://static.igem.org/mediawiki/2014/8/87/Mapa.png" style="width:100%;height:900px"/>
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<h1>Assembly Forms</h1>
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<h1>Assemblies forms</h1>
<p><strong>Assembly form template</strong></p>
<p><strong>Assembly form template</strong></p>
<p>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 :)</p>
<p>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 :)</p>
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<a href="https://static.igem.org/mediawiki/2014/e/eb/Assembly_Form.pdf"><img src="https://static.igem.org/mediawiki/2014/3/32/Assembly_form_pg3.png" style="width:700px;height:400px"/></a>
<a href="https://static.igem.org/mediawiki/2014/e/eb/Assembly_Form.pdf"><img src="https://static.igem.org/mediawiki/2014/3/32/Assembly_form_pg3.png" style="width:700px;height:400px"/></a>
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<h2>Main Assemblies<h2>
 
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<h2>Characterization Assemblies<h2>
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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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<h2>Promoter BBa_K823003</h2>
<p><strong>Question</strong>: Does the constitutive promoter BBa_K823003 work properly?</p>
<p><strong>Question</strong>: Does the constitutive promoter BBa_K823003 work properly?</p>
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<img src="https://static.igem.org/mediawiki/2014/7/7a/KXVI_w.png" width="600" height="350px"/>
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<img src="https://static.igem.org/mediawiki/2014/d/d5/KI_w.png" width="300" height="170px"/>
<p><strong>Results</strong>: After the incubation period of the transformed <em>E. coli</em> a large portion of the colonies were glowing green. So the promoter does work. Moreover, this biobrick works on <em>E. coli</em> despite the fact it was designed for <em>B. subtilis</em>.</p>  
<p><strong>Results</strong>: After the incubation period of the transformed <em>E. coli</em> a large portion of the colonies were glowing green. So the promoter does work. Moreover, this biobrick works on <em>E. coli</em> despite the fact it was designed for <em>B. subtilis</em>.</p>  
<img src="https://static.igem.org/mediawiki/2014/e/ef/Glowing_E_coli.JPG" width="400px" height="200px"/>
<img src="https://static.igem.org/mediawiki/2014/e/ef/Glowing_E_coli.JPG" width="400px" height="200px"/>
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<h2>Promoter BBa_K143015</h2>
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<p><strong>Question</strong>: How does the transcription caused by this promoter varies with the IPTG induction?</p>
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<img src="https://2014.igem.org/File:KXVI_w.png" width="700px" height="350px"/>
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<p><strong>Results</strong>:</p>
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<h2>Tunning of the QteE Threshold</h2>
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<p><strong>What are the concentration of QteE needed to hamper the LasR induction of the promoter PlasR?</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>
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<img src="https://static.igem.org/mediawiki/2014/8/86/KXV_w.png" width="700px" height="300px"/>
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<img src="https://static.igem.org/mediawiki/2014/c/c4/KXIII_w.png" width="700px" height="300px"/>
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<img src="https://static.igem.org/mediawiki/2014/4/43/KXIV_w.png" width="700px" height="300px"/>
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<h1>Assembly Forms<h1>
<table>
<table>

Revision as of 01:10, 9 October 2014


WELCOME TO iGEM 2014!

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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