Team:LCG-UNAM-Mexico:Journals:Uriel

Objectives
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 early lysis that occur when WT P4 and 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 by a promoter inducible by IPTG in conjunction with transactivators cox and ogr from phage P2. All this will 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.

Qualitative characterization of the multipromoter. The multipromoter that we have designed has the capacity to respond specifically to T3/T7 RNA polymerases so if one or both polymarease are presente in the cell the genes downstream of this promoter will be active. A first characterization approach is the introduction of a plasmid carring our promoter in the E. coli strain BL21(DE3)pLysS that has a T7 RNA polymerase inducible by IPTG

July 30, 2009
Primers for P2 cox and ogr genes arrived so we can start to construct the phage production system control.

To attain control of phage production we are going to put under an IPTG inducible promoter the genes that codify for the principal regulators of the morphopoietic genes and delete genes that doesn't belongs to the morphopoietic group this part will be done by Laura Gomez.

The genes that codify for this regulators are going to be amplified via colony PCR from the E. coli C-177 strain, which has a lysogenic form of phage 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 protocol and also de suffix and prefix iGEM sequences.

It is known that C-1a strain neither has a cox gene nor an ogr gene while C-1a K-12 strain contain a copy of ogr in its genome. We have performed a colony PCR over the next strains, looking for ogr and cox.

Genes: ogr cox

Strains: C-1a C-117 DH5alpha

July 31, 2009
Colony PCR is going to be done with strain C-1a and C-117 as a control DH5alpha which has 17, we are going to confirm that C-1a doesn't has a P2 by means of PCR for P2 cox and ogr genes.

Primers: Cox: >for_pre+rbs+cox_P2 GTTTCTTCGAATTCGCGGCCGCTTCTAGGGGGCTAGAGGACGACATGAG >rev_suf+dSTOP+cox_P2 GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAACGTGGTTCACCGAGAC

Ogr: >Forward_OGR GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCGGAAGAGGTGCTCGCGATG >Reverse_OGR GTTTCTTCCTGCAGCGGCCGCTACTAGTATTA,TTACATCCACATAATTTGCTGCCC

Expected Regions: Cox: Ogr:

Strains were platted on June 19, 2009 and were maintained at 4ºC and also a glycerol stock was created for C-1a and C-117. With a wood stick bacteria was taken and inoculated in LB without any selection.

The inoculated medium was incubated at 37ºC 250 rpm. for 1 hr. and then centrifuged at maximum speed with a tabletop centrifuge, supernatant was discarded and 200 µL of Tris-EDTA 10/1-NaCl 10mM added. The samples were heated over 10 min at 95ºC and then centrifuged again 2 min. 10µL of supernatant were taken for the PCR.

PCR Reaction: Water        24µL Buffer 10x   5µL MgCl2 50mM   2.5µL dNTP's 0.4mM 3.5µL Taq          1µL primer up    2.5µL primer low   2.5µL DNA          10µL Total        50µL

PCR results: Control		- C-117/ogr	+ C-1a/ogr	- DH5alpha/ogr	+ C-117/cox	+ C-1a/cox	- DH5alpha/cox	-

The PCR's were consistent with previous results in literature. For example ogr was positive for DH5alpha which is a derivative from K-12 and we could not obtain PCR products from C-1a. C-117 which has P2 in lysogenic state gave use positive PCR products for ogr and cox.

August 3, 2009
We prepared overnights of DH5alpha which contain the BioBricks pSB1AK3 and pSB1T3 the selective medium was 50µL/mL Kanamycin and 20µL/mL Tetracycline respectively

August 4, 2009


We redo the colony PCR for ogr and cox only using the C-117 strain. The PCR products were purified using the High Pure PCR Product Roche's Purification Kit and also plasmidic DNA was purified from the last overnights using the High Pure Plasmid Roche's Isolation Kit. The plasmids that were purified are going to used to add a double terminator sequence to ogr and clone cox for future manipulations.

1) ogr EcoRI/SpeI       2) cox EcoRI/PstI 3) BBa_B0015 EcoRI/XbaI (plasmid pSB1AK3)       4) BBa_J04450 EcoRI/PstI (plasmid pSB1T3)

Restriction Reactions: DNA	10µL Ezimas	.5µL each one Buffer	2µL Water	7µL Total	20µL

Vectors that will be use for cloning were dephosphorylated with Antarctic Phosphatase to avoid as much as possible false positives and also screening to many colonies.

Dephosphorylation Reaction: Plasmid		20µL Buffer		3µL Enzyme		1µL Water		6µL Total		30µL Incubation conditions: 1) 15 min --> 37ºC 	2) 5 min --> 65ºC

Parts were ligated in the following way:

1) ogr+BBa_B0015+pSB1T3 	2) cox+pSB1T3

Ligation Reactions: 1) 	Buffer	4µL 	Ligase	1µL 	pSB1T3	2µL 	ogr	6µL 	BBa_B0015	7µL 	Weter	0µL 	Total	20µL 	2) Buffer	4µL Ligase	1µL pSB1T3	3µL cox	6µL Weter	6µL Total	20µL The las reaction gave as a result the next parts:

1)ogr+BBa_B0015+pSB1T3         2)cox+pSB1T3

DH5alpha competent cells were transformed and platted on LB Agar with appropriate selection, for both constructions this selection was Tetracycline.

August 6, 2009
The ogr PCR product was cut with EbaI/PstI and cloned in pSB1T3 that was digested with the same enzymes and dephosphorylated as mentioned earlier. And then we performed a ligation reaction. The resulting BioBrick was named I-09#021.

1) ogr EcoRI/PstI

Restriction Reaction: DNA	10µL Ezimas	.5µL Buffer	2µL Water	7µL Total	20µL

A 1% agarose gel was prepared and run 1 hr. 85 V. to see the insert and plasmid concentration. Unfortunately we loose our ogr's PCR product so we performed a new PCR only for this gene.

August 7, 2009
The PCR for ogr was done using the DNA from strain C-117.

PCR Reaction: Water 	23µL Buffer	5µL MgCl2	2.5µL dNTPs 	.5	each Taq/pol	1µL Oligo	2.5	each DNA	10µL Total	50µL PCR: 1) Control	+ 	2) C-117/ogr	+ 3) C-117/ogr	+ 	4) C-117/ogr	+ 5) C-117/ogr	+ An Agarose gel was run and we find that one of our reactants was contaminated because our negative control was positive that PCR is going to be repeated but with different reactants

August 10, 2009
We are going to change the PCR reagents because we don't know if they are contaminated and perform the next PCR reaction.

Reagents to be changed: 1)primers for cox and ogr 2)buffer 10X 3)MgCl2

I'm going to do again the PCR for ogr and cox only from C-117 and as a control C-1a and DH5alpha the former control neither has a cox gene nor an ogr gene.

PCR Reactions:         1)     2)     3)     4)      5)      6)     7)      8)      9)     10) Water           23µL   +      +      +      +       +       +      +       +       +       + Buffer 10X       5µL   +      +      +      +       +       +      +       +       +       + MgCl2          2.5µL   +      +      +      +       +       +      +       +       +       + dNTP's         3.5µL   +      +      +      +       +       +      +       +       +       + Taq              1µL   +      +      +      +       +       +      +       +       +       + primer fw      2.5µL   +      +      +      +       +       +      +       +       +       + primer rev     2.5µL   +      +      +      +       +       +      +       +       +       + DNA tamplate    10µL   -      -      +      +       +       +      +       +       +       + 1)control for cox only primers 2)control for ogr only primers 3)control for DH5alpha ogr it must be positive 4)control for ogr & cox with strain C-1a it must be negative 5-7)PCR of interest positive for C-117 ogr gene 8-10)PCR of interest positive for C-117 cox gene

August 11, 2009


The PCR for ogr was done again and everything looked OK so we purified the PCR products with the High Pure PCR Product Roche's Purification Kit. An Agarose gel was run to see how much DNA was lost during the purification procedure and the using this information to add the right amount of restriction enzymes.

The first three lanes are cox, ogr and ogr+BBa_B0015 amplified using the prefix and suffix primer for these genes

Agust 13, 2009
Plasmid 18 was dephosphorylated because we are going to insert in it the ogr product that was amplified yesterday.

Dephosphorylation Reaction: Plasmid   20µL Buffer    3µL Enzyme    1µL Water     6µL Incubation: 1)15 min --> 37ºC    2)5 min  --> 65ºC

This plasmid that is going to be ligated with ogr is digested EcoRI/PstI.

cox was cut with EcoRI/SpeI and the plasmid that contain ogr+BBa_B0015 with EcoRI/XbaI because we are going to insert cox into this plasmid in order to concatenate cox+ogr+BBa_B0015.



August 14, 2009
Vector pSB1T3 was dephosphorylated and again a 1% Agarose gel was run with the vector and the ogr PCR product to check concentrations and posterior to this a ligation reaction was performed.

The Ligase enzyme that we are using is T4 DNA ligase from New England Biolabs which has the following reaction conditions

10 min at 23ºC and for inactivation incubate 20 min at 70ºC

Ligation Reaction: T4 Ligase	1µL Buffer		2µL ogr		2µL pSB1T3		5µL Water		10µL Total		20µL

Once the ligation reaction finished we transformed DH5alpha competent cells and platted them on selective medium.

August 18, 2009
We started to prepare glycerol for the BrioBricks and Strains that we have finished to store them at -70ºC.

The BBa_R0010/pSB1A2 which has an IPTG inducible promoter is going to be purified and digested to put under the control of this promoter the construction cox+ogr+BBa_B0015.

The plasmid was digested with SpeI/PstI and the insert with XbaI/PstI.

To construct cox+ogr+BBa_B0015 we took cox that was in pSB1T3 and ogr+BBa_B0015 also in pSB1T3 and perform the following reactions:

Restriction Reaction: Resistance pSB1T3+ogr+BBa_B0015	XbaI/PstI	 Tet pSB1T3+cox	       EcoRI/SpeI	  Tet pSB1C3		       EcoRI/PstI	  Cm

We expect white colonies and resistant to Cm because pSB1C3 has cloned an RFP protein that is expressed constitutively.

August 20, 2009
We performed restriction reactions with different colonies that carry the same plasmid to perform parallel constructions to avoid the case where a mutation could arise over one of the constructions leading this to the need of doing again the whole construction from a previous step.

DH5alpha strains that contain the listed plasmids below were grown in selective media and plasmid purification was performed and afterwards it was digested.

Plasmids: 1) ogr	XbaI/PstI 	2) ogr+pSB1CB	XbaI/PstI 3) ogr+pSB1CB	EcoRI/PstI 	4) ogr+pSB1CB	EcoRI/PstI 5) ogr+pSB1CB	EcoRI/PstI 	6) BBa_J04450	EcoRI/PstI 7) ogr+BBa_PB1005	EcoRI/PstI 	8) BBa_J04450	EcoRI/PstI 9) cox

Restriction Reactions: Buffer		4µL BSA		.4µL DNA		25µL Enzyme		2µL	each Water		6.6µL Total		40µL

August 21, 2009
An 1% Agarose gel was run for 1hr. at 85 V.

With this gel was confirmed that ogr is cloned in pSB1T3 and now we can make a glycerol for this strain.



First nine lanes interest.

24 Ago 2009
The pSB1C3 was dephosphorylated with antarctic phosphate

Dephospohrialtion Reaction: Plasmid		20µL Buffer		3µL Enzyme		1µL Water		6µL Total		30µL Incubation: 1) 15 min ––> 37ºC 		2) 5 min --> 65ºC

After plasmid dephosphorialtion we are going to perform a ligation reaction between cox and ogr+BBa_0015

August 25, 2009
Again an Agarose was run 1hr. at 85 V. to se the relative concentration of each sample that is going to be use for the ligation reaction.

Ligation Reaction: T4-ligase	 1µL Buffer		2µL #023.1		3µL #022.1		3µL #017.1		2µL Water		9µL Total		20µL Incubation: 1) 10 min. --> 20ºC-25ºC 		2) 20 min. --> 65ºC Controls: 1) Vector without dephosphorylation 		2) Dephosphorilated vector without insert

We platted DH5alpha transformed cells over selective medium and incubate the plates overnight at 37ºC.

August 27, 2009
Plasmid extraction



August 29, 2009
From ligation and transformation on Ago 25 a restriction analysis form tree selected proteins.

We are going to cut the purified plasmid from transformed cells in the following way:

1) I-09#012	SpeI/PstI 	2) I-09#24.1	XbaI/PstI 3) I-09#24.2	XbaI/PstI 	4) I-09#24.3	XbaI/PstI

Restriction Reaction: Buffer 		4µL BSA		0.4µL Enzyme		2µL each DNA		25µL Total		40µL We run a 1% Agarose gel at 85 V. 1hr



Unfortunately cox+ogr+BBa_B0015 cloning by fusing cox and ogr+BBa_B0015 in another plasmid didn't work out. We will try to repeat the procedure to see if we are luckier.

September 7, 2009
Again we dephophorylated vector I-09#017 to ligate it with cox and ogr+BBa_B0015 and then perform a transformation

Dephosphorylation Reaction: Plasmid		20µL Buffer		3µL Enzyme		1µL Water		6µL Total		30µL Incubation conditions: 1) 15 min --> 37ºC 	2) 5 min --> 65ºC

Ligation Reaction: T4-ligase	 1µL Buffer		2µL #023.1		3µL #022.1		3µL #017.1		2µL Water		9µL Total		20µL Incubation conditions: 1) 10 min. --> 20ºC-25ºC       2) 20 min. --> 65ºC Controls: 1) Vector without dephosphorylation        2) Dephosphorilated vector

We platted DH5alpha transformed cells over selective medium and incubated overnight at 37ºC.

September 11, 2009


Colony PCR reactions were done with selected colonies that resulted from the last transformation the resulting and tow of the twenty-one looked like they contained cox+ogr+BBa_B0015 verification with a restriction assay was done and also ogr+BBa_B0015+18 was purified and opened digested with EcoRI/PstI to be prepared in case last ligation don't work.

September 15, 2009
The ligation didn't work, I planned to follow a different strategy first purify the cox fragment using Low melt point Agarose, we are using this procedure because the plasmido that contain cox is the samen as the one that has ogr+BBa_B0015 and as a consequence the same antibiotic resistance gene, so by separating the DNA by electrophoresis we can take apart cox gene and the ligated in to the dephoshorylated plasmid ogr+BBa_B0015 that was digested EcoRI/XbaI, the resultin plasmid will be cox+ogr+BBa_B0015.

September 22, 2009
Band purification:

Quiagen gel exration kit was used for this.

An 1% Low melt point agarose gel was loaded with the sample that contained cox+18, the gel was stained with ethdium bromaid and saw with a transluminator. Once we saw our band it was cut from the gel and transferred to an 1.5 mL tube and weighted (0.13g) in the kit protocol they propose an equivalences of buffer 100mg~100µL so we have to add 3 volumes of each 130µL of buffer and follow the kit protocol.

September 23, 2009
A ligation is going to be done with cox and ogr+BBa_B0015, ogr+BBa_B0015+18 was dephosphorylated and cox was purified via gel. As a control we will try to religate the dephosphorylated plasmid, the same plasmid not dephosphorylated and a uncut plasmid which must transform because was not digested.

Ligation Reaction: T4 ligase  1µL Buffer     2µL ogr+BBa_B0015    2µL cox        5µL Water      10µL Total      20µL Controls: 1)dephosphorylated plasmid        2)non-dephosphorylated T4 ligase  1µL                    T4 ligase     1µL Buffer     2µL                    Buffer        2µL Plasmid    2µL                    Plasmid       2µL Water     15µL                    Water        15µL Total     20µL                    Total        20µL

Incubation: 1)10 min --> 25ºC    2)20 min --> 65ºC

DH5alpha competent cells were transformed and platted over selective media in this case Tet.

September 24, 2009
All controls resulted as expected so the must probable is that we achieved to ligate cox with ogr+BBa_B0015+18 I prepared overnights to extract plasmid tomorrow and perform a restriction assay to see if we have the fragment of interest.



September 25, 2009
To check last ligations a restriction assay is going to be done in a way that allows us to do the next ligation and finish the phage production control system.

Restriction Reactions: 1)XbaI/PstI cox+18             2)XbaI/PstI cox+ogr+BBa_B0015 colonies[1-6] Buffer 2      2µL              Buffer2           2µL BSA         0.2µL              BSA             0.2µL Water       6.8µL              Water           6.8µL DNA          10µL              DNA              10µL Enzyme      0.5µL each(X/P)    Enzyme          0.5µL each(X/P) Total        20µl              Total            20µL



As the image show on the first 7 lanes we were able to ligate cox with ogr+BBa_B0015+18 every of the selected clones has the cox insert so this procedure was faster than the fisr that we tried to use the only thing is that you need to purify the insert via an Agarose gel and the ligate it to the plasmid of interest.

We are ready for the last step, we are going to add the IPTG inducible promoter to get the following structure.

iptg+cox+ogr+ter

The second lane sample was selected for ligation with IPTG

Ligation Reaction: T4 ligase       1µL Buffer          2µL cox+ogr+BBa_B0015     5µL iptg            5µL Water          10µL Total          20µL

DH5alpha competent cells were transformed and platted over LB Amp100 and incubate at 37ºC

September 28, 2009
Controls were inconsistent in particular the re-ligation of dephosphorylated plasmid which gave the same amount of colonies compared with ligation of cox+ogr+BBa_B0015 and 12 so we are going to re-dephosphorylate the vector 12 and perform a new ligation reaction.

Dephosphorylation Reaction: Plasmid    10µL Buffer      3µL Enzyme      1µL Water      16µL Total      30µL Incubation 1)10 min --> 25ºC    2)20 min --> 65ºC

A 1% agarose gel was run to see the concetration of the plasmid and the insert, this to put the right amounts of insert and plasmid.

The samples that we are looking on the gel correspon to cox+ogr+BBa_B0015+18 digested with XbaI/SpeI and 12

Once we checked concetrations we can do the ligation reaction.

Ligation Reaction: T4 ligase    1µL Buffer       2µL cox+ogr+BBa_B0015  12µL 12           4µL Water        1µL Total       30µL

DH5alpha competent cells were transformed on LB agar Amp100 and incubated overnight 37ºC

September 29, 2009
Four colonies were selected an re-platted and also liquid 5 mL of LB was inoculated to prepare cultures for plasmid extraction.

September 30, 2009
An 1% Agarose gel was run to check concentrations for restriction assays and by this be able to know if we have the fragment we are interested.

The four plasmids were digested EcoRI/PstI

Restriction Reactions: Buffer    2µL BSA     0.2µL DNA      10µL Enzyme    1µL each  EcoRI/PstI Water   5.8µL



The insert looks shifted upwards but a little amount so we didn't achieve to ligate the iptg to cox+ogr+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.

Going back further when I digested for the first time plasmid pSB1A2 I did it with SpeI/PstI to allow promoter to be at the beginning of the construction.The insert cox+ogr+BBa_B0015 was digested XbaI/PstI so I think the unique alternative is that promoter ipteg wasn't in the plasmid.

In this circumstance we can only say that we changed cox+ogr+BBa_B0015 from plasmid pSB1T3 to pSB1A212 that is resitantant to Amp.

October 5, 2009
To test for the activity of the multipromoter that we designed and synthesized, we are going to transform E. coli 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.

The IPTG preparation protocol we used is discribed 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 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



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
I prepared cultures to purify plasmid that contains the parts that we are planning to send to the registry.

Part List: cox ogr ogr+cox+BBa_B0015 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.

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 References.