Team:LCG-UNAM-Mexico:Journals:Uriel

31 Jul 2009

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.

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 e 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 ogr gene while 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.

C-1a C-117 DH5alpha

PCR Reaction:

PCR: 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.

3 Ago 2009

We prepared overnights of DH5alpha which contain the BioBricks I-09#005 and I-09#018 the selective medium was 50µL/mL Kanamycin and 20µL/mL Tetracycline respectively

4 Ago 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) I-09#005 EcoRI/XbaI (plasmid) 4) I-09#018 EcoRI/PstI (plasmid)

Restriction Reactions:

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

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

Dephosphorylatio Reaction:

Se ligaron de la siguiente forma.

1) ogr+I-09#005+I-09#018 2) cox+I-09#018

Ligation Reactions:

1)

Buffer 4µL Ligase 1µL #018 2µL ogr 6µL #005 7µL Weter 0µL Total 20µL

2)

Buffer 4µL Ligase 1µL #018 3µL cox 6µL Weter 6µL Total 20µL

The las reaction gave as a result the next BioBricks

1)ogr+I-09#005+I-09#018 --> I-09#023 2)cox+I-09#018 --> I-09#022

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

6 Ago 2009

The ogr PCR product was cut with EbaI/PstI and cloned in I-09#018 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.

7 Ago 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

10 Ago 2009

The PCR for ogr was done again and everything looked OK so we purified the PCR product 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.

14 Ago 2009

Vector I-09#018 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 #018 5µL Water 10µL Total 20µL

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

18 Ago 2009

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

The I-09#012 which has an IPTG inducible promoter is going to be purified and digested to put under the control of the IPTG promoter the cox+ogr+ter construction.

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

To construct cox+ogr+ter we took cox that was in I-09#018 and ogr+ter also in I-09#018 and perform the following reactions:

Restriction Reaction: Resistance #018+ogr+ter XbaI/PstI Tet #018+cox EcoRI/SpeI Tet #017 EcoRI/PstI Cm

We expect white colonies and resistant to Cm because I-09#017 has cloned an RFP protein that is expressed constitutively.

20 Ago 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) I-09#023.1 XbaI/PstI 2) I-09#022.1 XbaI/PstI 3) I-09#017.1 EcoRI/PstI 4) I-09#017.2 EcoRI/PstI 5) I-09#021.1 EcoRI/PstI 6) I-09#021.2 EcoRI/PstI 7) I-09#021.3 EcoRI/PstI 8) I-09#021.4 EcoRI/PstI

Restriction Reactions:

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

21 Ago 2009

An 1% Agarose gel was run for 1hr. at 85 V.

With this gel was confirmed that ogr is cloned in I-09#018 and now we can make a glycerol for this strain.

24 Ago 2009

The I-09#017 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 I-09#022.1 and I-09#023.1

25 Ago 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.

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

7 Sep 09

Again we dephophorylated vector I-09#017 to ligate it with cox and ogr+ter 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 incubate the plates overnight at 37ºC.

11 Sep 09

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+ter verification with a restriction assay was done and.