Team:LCG-UNAM-Mexico:Journals:Uriel
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Construction of the phage production control system. | Construction of the phage production control system. | ||
- | In order to produce a grate amount of P4 phage particles that has our death system, we want to avoid the natural | + | In order to produce a grate amount of [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P4] phage particles that has our death system, we want to avoid the natural |
- | early lysis that occur when WT P4 and P2 interact. This avoidance will be achieved by taking the control of the | + | early lysis that occur when WT [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P4] and [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2] interact. This avoidance will be achieved by taking the control of the |
- | two major regulators of P2 morphopoietic genes. The control systems that is going to be implemented is constituted | + | two major regulators of [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2] morphopoietic genes. The control systems that is going to be implemented is constituted |
- | by a promoter inducible by IPTG in conjunction with transactivators [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr] from phage P2. All this will | + | by a promoter inducible by IPTG in conjunction with transactivators [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr] from phage [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2]. All this will |
allow us to grow bacteria in grate quantities and induce lysis when ever we want and as a consequence the | allow us to grow bacteria in grate quantities and induce lysis when ever we want and as a consequence the | ||
- | production of grate amounts of P4 phage. | + | production of grate amounts of [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P4] phage. |
- | Qualitative characterization of the multipromoter. | + | Qualitative characterization of the [http://partsregistry.org/Part:BBa_K242100 multipromoter]. |
The multipromoter that we have designed has the capacity to respond specifically to T3/T7 RNA polymerases | The multipromoter that we have designed has the capacity to respond specifically to T3/T7 RNA polymerases | ||
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The genes that codify for this regulators are going to be amplified via colony PCR from the E. coli [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-177] strain, which | The genes that codify for this regulators are going to be amplified via colony PCR from the E. coli [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-177] strain, which | ||
- | has a lysogenic form of phage P2. The primers | + | has a lysogenic form of phage [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2]. The primers |
that we are using amplify the genes with its WT RBS's and also introduce an extra stop codon as required by the standardization | that we are using amplify the genes with its WT RBS's and also introduce an extra stop codon as required by the standardization | ||
protocol and also de suffix and prefix iGEM sequences. | protocol and also de suffix and prefix iGEM sequences. | ||
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Colony PCR is going to be done with strain [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-1a] and [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-117] as a control [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] which has 17, we are going | Colony PCR is going to be done with strain [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-1a] and [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-117] as a control [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] which has 17, we are going | ||
- | to confirm that [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-1a] doesn't has a P2 by means of PCR for P2 [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr] genes. | + | to confirm that [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-1a] doesn't has a [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2] by means of PCR for [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2] [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr] genes. |
Primers: | Primers: | ||
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The PCR's were consistent with previous results in literature. For example | The PCR's were consistent with previous results in literature. For example | ||
[http://partsregistry.org/Part:BBa_K242001 ogr] was positive for [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] which is a derivative from [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains K-12] and we could | [http://partsregistry.org/Part:BBa_K242001 ogr] was positive for [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] which is a derivative from [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains K-12] and we could | ||
- | not obtain PCR products from [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-1a]. [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-117] which has P2 in lysogenic state gave use positive PCR products for [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242002 cox]. | + | not obtain PCR products from [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-1a]. [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-117] which has [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains P2] in lysogenic state gave use positive PCR products for [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242002 cox]. |
== August 3, 2009 == | == August 3, 2009 == | ||
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== August 4, 2009 == | == August 4, 2009 == | ||
+ | [[Image:5-ago-dr para ligar.jpg|200px]] | ||
We redo the colony PCR for [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242002 cox] only using the [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-117] strain. The PCR | We redo the colony PCR for [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242002 cox] only using the [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains C-117] strain. The PCR | ||
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The las reaction gave as a result the next parts: | The las reaction gave as a result the next parts: | ||
- | 1)[http://partsregistry.org/Part:BBa_K242001 ogr]+ | + | 1)[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]+[http://partsregistry.org/Part:pSB1T3 pSB1T3] |
2)[http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:pSB1T3 pSB1T3] | 2)[http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:pSB1T3 pSB1T3] | ||
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the using this information to add the right amount of restriction enzymes. | the using this information to add the right amount of restriction enzymes. | ||
- | The first three lanes are [http://partsregistry.org/Part:BBa_K242002 cox], [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 amplified using the prefix and suffix | + | The first three lanes are [http://partsregistry.org/Part:BBa_K242002 cox], [http://partsregistry.org/Part:BBa_K242001 ogr] and [http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] amplified using the prefix and suffix |
primer for these genes | primer for these genes | ||
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This plasmid that is going to be ligated with [http://partsregistry.org/Part:BBa_K242001 ogr] is digested EcoRI/PstI. | This plasmid that is going to be ligated with [http://partsregistry.org/Part:BBa_K242001 ogr] is digested EcoRI/PstI. | ||
- | [http://partsregistry.org/Part:BBa_K242002 cox] was cut with EcoRI/SpeI and the plasmid that contain [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 with EcoRI/XbaI because we are | + | [http://partsregistry.org/Part:BBa_K242002 cox] was cut with EcoRI/SpeI and the plasmid that contain [http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] with EcoRI/XbaI because we are |
going to insert [http://partsregistry.org/Part:BBa_K242002 cox] into this plasmid in order to concatenate [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015. | going to insert [http://partsregistry.org/Part:BBa_K242002 cox] into this plasmid in order to concatenate [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015. | ||
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[https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] strains that contain the listed plasmids below were grown in selective media and plasmid | [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] strains that contain the listed plasmids below were grown in selective media and plasmid | ||
purification was performed and afterwards it was digested. | purification was performed and afterwards it was digested. | ||
+ | |||
+ | |||
Plasmids: | Plasmids: | ||
- | 1) | + | 1) ogr XbaI/PstI |
- | 2) | + | 2) ogr+pSB1CB XbaI/PstI |
- | 3) | + | 3) ogr+pSB1CB EcoRI/PstI |
- | 4) | + | 4) ogr+pSB1CB EcoRI/PstI |
- | 5) | + | 5) ogr+pSB1CB EcoRI/PstI |
- | 6) | + | 6) BBa_J04450 EcoRI/PstI |
- | 7) | + | 7) ogr+BBa_PB1005 EcoRI/PstI |
- | 8) | + | 8) BBa_J04450 EcoRI/PstI |
+ | 9) cox | ||
Restriction Reactions: | Restriction Reactions: | ||
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With this gel was confirmed that [http://partsregistry.org/Part:BBa_K242001 ogr] is cloned in [http://partsregistry.org/Part:pSB1T3 pSB1T3] and now we can make a glycerol for this strain. | With this gel was confirmed that [http://partsregistry.org/Part:BBa_K242001 ogr] is cloned in [http://partsregistry.org/Part:pSB1T3 pSB1T3] and now we can make a glycerol for this strain. | ||
- | 24 Ago 2009 | + | [[Image:21-08-09--ogrpcr-4xogr+18-17-cox-17-ogr+ter.jpg|200px ]] |
+ | |||
+ | First nine lanes interest. | ||
+ | |||
+ | == 24 Ago 2009 == | ||
- | The | + | The pSB1C3 was dephosphorylated with antarctic phosphate |
Dephospohrialtion Reaction: | Dephospohrialtion Reaction: | ||
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2) 5 min --> 65ºC | 2) 5 min --> 65ºC | ||
- | After plasmid dephosphorialtion we are going to perform a ligation reaction between | + | After plasmid dephosphorialtion we are going to perform a ligation reaction between cox and ogr+BBa_0015 |
== August 25, 2009 == | == August 25, 2009 == | ||
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We platted [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] transformed cells over selective medium and incubate the plates overnight at 37ºC. | We platted [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains DH5alpha] transformed cells over selective medium and incubate the plates overnight at 37ºC. | ||
+ | |||
+ | == August 27, 2009 == | ||
+ | |||
+ | Plasmid extraction | ||
+ | |||
+ | [[Image:3x024-pcrcontrol-x007.jpg|250px]] | ||
== August 29, 2009 == | == August 29, 2009 == | ||
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We run a 1% Agarose gel at 85 V. 1hr | We run a 1% Agarose gel at 85 V. 1hr | ||
+ | |||
+ | [[Image:Ogr+ter-cox-17dfos.jpg|250px]] | ||
Unfortunately [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 cloning by fusing [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 in another plasmid | Unfortunately [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 cloning by fusing [http://partsregistry.org/Part:BBa_K242002 cox] and [http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 in another plasmid | ||
didn't work out. We will try to repeat the procedure to see if we are luckier. | didn't work out. We will try to repeat the procedure to see if we are luckier. | ||
- | |||
== September 7, 2009 == | == September 7, 2009 == | ||
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- | The samples that we are looking on the gel correspon to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015+18 digested with XbaI/SpeI and 12 | + | The samples that we are looking on the gel correspon to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]+18 digested with XbaI/SpeI and 12 |
Once we checked concetrations we can do the ligation reaction. | Once we checked concetrations we can do the ligation reaction. | ||
Line 654: | Line 669: | ||
T4 ligase 1µL | T4 ligase 1µL | ||
Buffer 2µL | Buffer 2µL | ||
- | [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 12µL | + | [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] 12µL |
12 4µL | 12 4µL | ||
Water 1µL | Water 1µL | ||
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[[Image:12oct09-iptg-cox+ogr+ter-final.jpg|200px]] | [[Image:12oct09-iptg-cox+ogr+ter-final.jpg|200px]] | ||
- | The insert looks shifted upwards but a little amount so we didn't achieve to ligate the iptg to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 | + | The insert looks shifted upwards but a little amount so we didn't achieve to ligate the iptg to [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] |
this is strange because the plasmid maintained its size so the promoter is not there.I've checked the plasmid size an it is 2079 bp so the plasmid doesn't has the promoter. | this is strange because the plasmid maintained its size so the promoter is not there.I've checked the plasmid size an it is 2079 bp so the plasmid doesn't has the promoter. | ||
- | Going back further when I digested for the first time plasmid | + | Going back further when I digested for the first time plasmid [http://partsregistry.org/Part:pSB1A2 pSB1A2] I did it with SpeI/PstI to allow |
- | promoter to be at the beginning of the construction.The insert [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 was digested XbaI/PstI so I think the unique alternative is that promoter ipteg wasn't | + | promoter to be at the beginning of the construction.The insert [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] was digested XbaI/PstI so I think the unique alternative is that promoter ipteg wasn't |
in the plasmid. | in the plasmid. | ||
- | In this circumstance we can only say that we changed [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+BBa_B0015 from plasmid | + | In this circumstance we can only say that we changed [http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] from plasmid [http://partsregistry.org/Part:pSB1T3 pSB1T3] to [http://partsregistry.org/Part:pSB1A212 pSB1A212] that is resitantant to |
Amp. | Amp. | ||
== October 5, 2009 == | == October 5, 2009 == | ||
- | To test for the activity of the multipromoter that we | + | To test for the activity of the [http://partsregistry.org/Part:BBa_K242100 multipromoter] that we designed and synthesized, we are going to |
- | transform E. coli [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains#Expression_Strains BL21(DE3)plysS] | + | transform E. coli [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains#Expression_Strains BL21(DE3)plysS] from novagen. This strain uses a T7 phage polymerase that is controlled thorough Lac promoter. The multipromoter is composed of T7 & T3 promoters, we expect that our construction will respond to IPTG. |
- | This strain uses a T7 phage polymerase that is controlled | + | |
- | + | The IPTG preparation protocol we used is discribed [http://openwetware.org/wiki/IPTG HERE!!!] | |
- | + | ||
- | + | Transformed cells were platted on LB medium with Kn and IPTG .5 mM and expect that the colonies will fluoresce | |
+ | and could be seeing with a transluminator. | ||
- | + | == Octuber 6, 2009 == | |
- | + | We took a look to the LB Agar plates with a transluminator at a wavelength of 395nm but because we didn't have a | |
- | + | filter for GFP emitting wavelength we couldn't discriminate for our wavelength of interest. | |
- | + | We are going to try other approach this time with a microscope more suitable to see GFP. | |
- | + | ||
- | + | == October 7, 2009 == | |
+ | |||
+ | REGISTRY SITE FOR [http://partsregistry.org/wiki/index.php?title=Part:BBa_K242101 MULTIPROMOTER] BBa_K242101 | ||
+ | |||
+ | m stands for multipromoter !! | ||
+ | |||
+ | Cultures were prepared as follows: | ||
+ | |||
+ | BL21/m+ BL21/m+ BL21/m- | ||
+ | 100 mL LB + + + | ||
+ | 100 µL Kan 30 µg/µL + + - | ||
+ | 100 µL Cam 20 µg/µL + + + | ||
+ | 10 µL IPTG 1M + - + | ||
+ | |||
+ | |||
+ | For the induction of protein production we started our culture at .1 abs 620nm in 100mL LB and waited until it reached 0.35 abs 620 nm then we added IPTG to a final concentration of 0.1mM and then we leave the culture media for four | ||
+ | hours. | ||
+ | |||
+ | 5mL of each culture were centrifuged at max speed and the supernatant was discarded. We prepared our samples | ||
+ | to see them. Using a microscope with suitable filters and light to see GFP we compared the cultures between them | ||
+ | |||
+ | Results: | ||
+ | Bl21/m+ Bl21/m+ Bl21/m- | ||
+ | IPTG + - + | ||
+ | ++++ ++ - Fluorescence | ||
[[Image:Gfp multi.JPG |280px]] | [[Image:Gfp multi.JPG |280px]] | ||
- | Great NEWS!!! our promoter worked for T7 polymerase | + | In frame is BL21/multipromoter with IPTG inducer at 100X. Unfortunately the microscope and camera were not suitable to check in full detail BL21/m- IPTG+, which gave no fluorescence, and Bl21/multipromoter IPTG-, which presented a diminished fluorescence. |
+ | |||
+ | This results support the functioning of multipromoter for T7 polymerase. | ||
+ | |||
+ | Great NEWS!!! our promoter worked for T7 RNA polymerase | ||
+ | |||
+ | For future experiments to get a better characterization look result site. | ||
== October 20, 2009 == | == October 20, 2009 == | ||
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[http://partsregistry.org/Part:BBa_K242002 cox] | [http://partsregistry.org/Part:BBa_K242002 cox] | ||
[http://partsregistry.org/Part:BBa_K242001 ogr] | [http://partsregistry.org/Part:BBa_K242001 ogr] | ||
- | [http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_K242002 cox]+BBa_B0015 | + | [http://partsregistry.org/Part:BBa_K242001 ogr]+[http://partsregistry.org/Part:BBa_K242002 cox]+[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] |
- | + | [http://partsregistry.org/Part:BBa_K242100 multipromoter] | |
- | Three overnights for multipromoter testing were prepared, one would be induced by IPTG, other not and the latter is a BL21 strain that doesn't have the construction but will be grown with IPTG. | + | Three overnights for [http://partsregistry.org/Part:BBa_K242100 multipromoter] testing were prepared, one would be induced by IPTG, other not and the latter is a BL21 strain that doesn't have the construction but will be grown with IPTG. |
== October 21, 2009 == | == October 21, 2009 == | ||
+ | Preparation of parts for sending them to the registry!!! | ||
+ | =References= | ||
+ | To see all the literature that we used to develop the construction of control system pleas visit | ||
+ | references site and look for ogr, cox, P2 and P4 papers [https://2009.igem.org/Team:LCG-UNAM-Mexico/Description#References References]. | ||
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Latest revision as of 03:48, 22 October 2009