Team:UNAM Genomics Mexico/Notebook/CompleteNotebook
Our group is in charge of building part of the ìandî construction. We started analyzing if the plasmid we have with P4 actually had what we needed.
The plasmid PRMn25 contains the protein P4. It has Amp100 resistance and comes in Escherichia coli NFI. Cells were lysed to make sure the plasmid was present in these cells (5000bp). We ran a gel with 3 lysis, a sample from the plasmid PFRC54 (A3 promoter) and a sample of total DNA from the strain from which PFRC54 was obtained.
Plasmid PRMn25 was digested with SpeI and EcoRI. We do not have the sequence of the plasmid, but there seem to be several restriction sites for SpeI and just one site for EcoRI. The digestions were left for 4 hours and they were plused in the microwave 3 times, 10 seconds each time. Only Dulceís digestion worked, even though it was the ìdirtiestî sample.
We repeated the lysis of PRMn25.
Digestions (20 µl)
H2O 12 µl
Enzime 1 µl
Buffer 10x 2 µl
Plasmid 5 µl
PRMn25 was digested with EcoRi and BamHI. Since we donít have the sequence we used these enzyme to confirm the restriction sites mentioned in Rojo et al. (1990). These sites were corroborated and the vector was linearized. We recycled the gel used for the PRMn25 lysis, which is why the gel seems out of phase.
PY BROTH 10g salt/L
We transformed RFP E1010.
plate 1 18F
plate 2 17E
Stock 50 mg/mL
We made liquid cultures with colonies LIQUID CULTURE.
We extracted plasmids PLASMID EXTRACTION PROTOCOL.
We ran a gel to check the extraction GEL ELECTROPHORESIS PROTOCOL.
The lysis worked so we transformed PRMn25 in DH5a. We performed a lysis on these cells to see if they had been transformed correctly with PRMn25.
We also transformed PFRC54. TRANSFORMATION PROTOCOL
We designed primers for our project. (Note from 07/10/12: we redesigned the primer for LasR, since the sequence was incorrect.)
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCATTAGTAGAT
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga 3'
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta
ARAC without LVA (version 2 registry part: BBa_C0080)
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA
We extracted from gel LIQUID CULTURE.
We made glycerols of pHp45 O GLYCEROL PROTOCOL .
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450 PSB4A5 Am (ampicillin) 1I BBa_J04450 AraC BBa_C0080
2012 14L plate 1 pSB2K3 Km+
2011 14L plate 1 pSB2K3 Km+
2012 14L plate 1 pSB2K3 Km+
We obtained the terminator from the 2012 distribution (plate 2 24C) and we transformed the DNA in DH5a TRANSFORMATION PROTOCOL. The terminator (BBa_B0014) comes in the PSBIAK3 plasmid with resistance to Amp and Km. The RFP (BBa_E1010) comes in the plasmid PSBI2K3 with resistance to Amp and we obtained it from the 2012 distribution plate 2 (17E). We obtained the purified plasmid of the RFP from the AraC AND team. (Diego, Jonathan, and Abiel).
Due to the failed digestions, we did the RFP and terminator lysis again.
The AraC team has the terminator plasmid purified, we are thinking of using that one.
> Digested pHp45 O with E/P LIQUID CULTURE.
> We extracted plasmid with kit.
> Ran a gel GEL ELECTROPHORESIS PROTOCOL . (5)
>Extracted from gel LIQUID CULTURE.
>From the overnight plate we prepared liquid culture LIQUID CULTURE.
>We left them incubating overnight.
>We left a plate (LB Km DH5a C0080 and a control).
>Plasmid extraction AraC with kit.
>C0080-psB2K3 915 bp
>Streaked 4 LB Km 30 plates DH5a psB2K3.
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red.
>Ran gel with Spr/Strr (eppendorf -20∫C) GEL ELECTROPHORESIS PROTOCOL.
>Made glycerols from one tube of DH5a C0080 Km 50 -> 04.
>Made glycerols from one tube of DH5a PSB4A5 Amp 100 -> 05 GLYCEROL PROTOCOL.
>Digested C0080 X,S.
>Digested B0014 E,P LIQUID CULTURE.
We used the RFP that we transformed and the RFP that the AraC team did as a positive control. The terminator still looks strange, since we canít observe supercoling which is normally observed.
We are waiting for our primers, and the team will be going to a math modeling course for a week.
>Digested B0014 with E, P and with E,X. LIQUID CULTURE.
>Ran gels with yesterdayís digestions GEL ELECTROPHORESIS PROTOCOL .
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation.
C0080 is in the first lane.
>Extracted from gel C0080 X,S LIQUID CULTURE.
>Made glycerols from 4 plates LB Km DH5a pSB2K3 GLYCEROL PROTOCOL .
>Take out AmyE 5í from distribution [DNA KIT PLATE INSTRUCTIONS].
AmyE 5í 18K plate 3 2010, 2011, 2012
AmyE 3í 18M plate 3 2010, 2011, 2012
>Digested B0014 with E,X and E,P again LIQUID CULTURE.
>Ran gels with digestions B0014 with E,X and E,P GEL ELECTROPHORESIS PROTOCOL.
>Dephosphated B0014 E,X and B0014 E,P [DESPHOPHORYLATION PROTOCOL].
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS].
>Ran gel with psB2K3 and psB4A5 GEL ELECTROPHORESIS PROTOCOL .
>Transformed with plasmid B0079 1576 bp psB1A2 12A TRANSFORMATION PROTOCOL.
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011
AmyE 5í BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011
AmyE 3í BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011
AmyE 5í grew 2 colonies.
>We did a DH5a K143001 Km30 Amp 100 glycerol GLYCEROL PROTOCOL 07.
>We plated twice DH5a K143002 Km 30 Amp 100 + control and twice DH5a B0079 Amp 100 + control
These were both plated in 2 plates each.
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made.
>Liquid cultures LIQUID CULTURE.
2 tubes DH5a K143001 Km30 Amp 100
2 tubes DH5a K143002 Km30 Amp 100
2 tubes DH5a B0079 Amp 100
1 tube LB Km 30 Amp 100 control
1 tube LB Amp 100 control
>From the 6 tubes we extracted plasmid from kit.
We did 2 PCRs, since the primers have arrived. We purified by miniprep RFP and P4. With this PCR we will add the prefix (EcoRI and XbaI) and RBS to the beginning of the sequence, and suffix (restiction sites for SpeI asn PstI).
RFP, P4 PCR PROTOCOL
RFP UP 78∫C
RFP LW 75.5 ∫C
P4 UP 73.6 ∫C
P4 LW 73.9∫C
Taking into account both TMís, we used the same amplification program for both, thermocycle B progam iGEM.
We ran a gel and we observed fragments that correspond to the RFP and P4, we can also observe other fragments which means we will have to purify the bands.
We used Roche kit for band purification.
The P4 purification turned out slightly dirty and the RFP turned out completely dirty. We repeated the purifications by loading the previously ìpurifiedî sample and repeating the procedure.
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL].
>Ligated Cassete OSpr/Strr +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL].
>Transformed ligation and left overnight plated TRANSFORMATION PROTOCOL.
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
>Digested with PstI LIQUID CULTURE.
We repeated the gel where we checked the re-purification, and indeed we need to repeat P4ís PCR.
The PCR worked, but a band purification will be needed. We fused all of the PCR product and loaded it.
We grew bacterial culture in antibiotics for LasR lysis.
Rfp+terminator ligation LIGATION PROTOCOL
Cells were transformed with DH5a with RFP+terminator ligation.TRANSFORMATION PROTOCOL
LasR lysis were digested. We ran a gel with LasR PCRs to add RBS prefix and suffix.
We ran LasRís PCRs again, the last PCR didnít work, which is why we lowered the temperature to 65∫C. We left the digestions all night. PCR PROTOCOL
Since they didnít work, we decided to do it directly from the distribution from 2012 and 2010.
We noticed our mistakeÖ. The sequence we designed our primers for was an incorrect sequence!
We need to redo LasR primers.
Our ligation didnít work.
We redid RFP+terminator ligation and left it for the weekend. LIGATION PROTOCOL
4 LB Km30 Spec60 DH5a OSpr/Strr +K143002 still have not grown.
Ran a gel with yesterdayís digestions to chek if they were done properly (10)
GEL ELECTROPHORESIS PROTOCOL .
Ran another gel with the rest of the samples.
Extracted GusA fragment
Repeated ligation OSpr/Strr +K143002 E,X dephosphated LIGATION PROTOCOL.
Repeated OSpr/Strr PCR.
Transformed DH5a OSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5a OSpr/Strr +K143002 .
Transformed DH5a OSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5a OSpr/Strr +K143002.
From yesterdayís transformations only one colony grew.
From the previous transformation only 2 colonies grew.
These 3 were streaked in 3 plates:
DH5a OSpr/Strr +K143002 LB Km 30 Sp 100 1.
DH5a OSpr/Strr +K143002 LB Km 30 Sp 100 2.
DH5a OSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km 30 Sp 100 control.
Did liquid cultures in 3 tubes:
OSpr/Strr +K143002 LB Km 30 Sp 100 1.
OSpr/Strr +K143002 LB Km 30 Sp 100 2.
OSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km30 Sp 100 control.
Ran a gel with: (11)
O E PCR P
O PCR P
O PCR I
O PCR I
GEL ELECTROPHORESIS PROTOCOL
Did GusA primers dissolution for PCR.
Gel Extraction by kit of lanes 3 and 5 LIQUID CULTURE.
PCR omega P, PCR omega I, PCR AraC P, PCR AraC I
Add 10 µl of each primer (LW and UP).
Add 3 µl of plasmid (P).
Add 30.4 µl buffer.
Add 5 µl Mg.
Add 8 µl DNTpís.
Add 42.6µl H2O miliQ.
Add 1 µl RTTG polymerase.
Centrifuge (spin) 8 secs.
Add vegetable oil till the eppendorf is full.
Place eppendorf 1 mL in thermocycler.
Run PCR with program ìBERNAî.
By 9am we didnít observe colonies in our LB Km30 with yesterdayís transformation, so using yesterdayís RFP digestions we redid the ligation.
We did another PCR of RFP because we ran out of it.
We ran a gel with the dephosphated terminator and digested RFP.
The PCR turned out ok except for some non-specific bands, we will need to extract the correct DNA band.
ïGusA digestions with X,P; O+AmyE 3í with E,X; AraC PCR with E,S (2 times) DIGESTIONS. (11.2)
We loaded the whole PCR product from the PCR.
>We took a picture of the gel to check the purification elutions but we did not observe anything so we quantified it on the nanodrop.
Sample 1) 5.9 ng/µl
Sample 2) 2.5 ng/µl
>With the RFP digested with EcoRI and SpeI we did another ligation with the terminator digested with EcoRI and XbaI and left it at 22∫C.
>We transformed yesterdayís ligation in DH5a and plated it in LB Km30. Since our ligations have not worked we decided to redo the digestions on the terminator (E0014). We asked the other AND team to give us the purified plasmid, but it wasnít enough so we streaked 2 plates LB Km30 so we could have the strain with the terminator. Tomorrow we will put liquid cultures and do lysis.
>We did digestions in another vector to see the efficiency of the enzymes as a test. We used PBBR-GusA with 5 µl of PBBR GusA, one digestion with 1.5 µl of EcoRI, one with 1 µl of SpeI and one with 1 µl of SpeI and 1.5 µl of EcoRI.
We left them at 37∫C.
>We did 5 new RFP PCRs.
>We streaked again the following strains:
The PCRs seem to show the band we need, we will have to extract them.
We checked the transformations from the ligation ì3î from RFP+terminator. We didnít observe colonies.
>We transformed the ligation ì4î RFP+terminator in DH5a, they were plated in LB Km30 .
>We ran a gel with GusA digestions.
>We digested 80 µl of the purified RFP with EcoRI and SpeI DIGESTION PROTOCOL.
>We left liquid cultures in LB Km30 so we can do an alkaline lysis of the plasmid containing the terminator (B0014).
ïRan a gel with yesterdayís digestions: (12)
ïTransformed AraC PCR+ O+AmyE 3í ligation in two tubes TRANSFORMATION PROTOCOL.
ïFrom pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïMade glycerols from 2 LB Km 30 Sp100 DH5a O+AmyE 3í plates and from 1 LB Amp100 pfrc54 (A3) plates with control GLYCEROL PROTOCOL .
ïLigated GusA PCR with B0014 E,X desphophorylated LIGATION PROTOCOL.
ïDigested Pfrc54 (A3) with S,P LIQUID CULTURE.
ïDesphophorylated O+AmyE 3í E,X [DESPHOPHORYLATION PROTOCOL].
ïRan gel with pfrc54 S,P GEL ELECTROPHORESIS PROTOCOL . (13)
ïTransformation of GusA PCR + B0014 ligation TRANSFORMATION PROTOCOL.
ïTransformed with GFP E0040 psBIA2.
ïLiquid Cultures with ligation: AraC+ O+AmyE 3í for plasmid extraction at night TRANSFORMATION PROTOCOL.
ïB. Subtitils competent cells [B.Subtilis group protocol].
>We did alkaline lysis of the terminator cultures from last night. We ran it and they all look ok, but weíll use the first one because it seems to be the cleanest.
We ligated RFP/terminator and ran a gel to see RFP and the terminator. LIGATION PROTOCOL.
>We checked the transformations and didnít see colonies.
ïFrom 24 AraC+ O+AmyE 3í LB Km 30 Sp 100 DH5a tubes:
ïExtracted plasmids [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïRan gel GEL ELECTROPHORESIS PROTOCOL .
ïFrom transformed DH5a km 30:
GusA+B0014 DH5a Km50 24 pellets
E0040 DH5a LB Amp100
From these two we:
ïDid liquid cultures LIQUID CULTURE.
ïExtracted plasmid [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïFrom the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid LIQUID CULTURE.
ïRan gel with this transformation GEL ELECTROPHORESIS PROTOCOL .
ïDigested E,P AraC+ O+AmyE 3í
ïO+AmyE 3í E,P LIQUID CULTURE.
We ran a gel with the RFP digestion, the purified terminator, and the RFP terminator ligation that was left for the whole weekend. (GEL NOT SHOWN)
>We digested the terminator with EcoRI and XbaI DIGESTION PROTOCOL.
> We digested RFP 1 with EcoRI and SpeI DIGESTION PROTOCOL.
In the gel we can see that the terminator digestion in ok, although it is still partial. We need to leave it for the whole night. We canít see the RFP digestion; it is possible that it degraded. We will concentrate RFP.
We ran 6 50 µl reactions of the PCR to amplify RFP. We will use as DNA RFP from the lysis, one from 2012, and one from 2010. We will make a dilution, use 1 µl of lysis and diluted them in H2O.
>We can still observe bands in the purification, we will try to do a better PCR.
>We digested the terminator lysis with EcoRI and XbaI and we decided to switch buffers. We used buffer 2 even though EcoRI might have a star effect. DIGESTION PROTOCOL
ïRan gel with yesterdayís digestions LIQUID CULTURE. (14)
ïThe gel we ran didnít work, probably because the agarose was not prepared correctly.
ïDigested 12, 15, 18, 21 with AraC+ O+AmyE 3 with E and also with Pí; B0014 with E,X; E0040 with X,S LIQUID CULTURE.
ïFrom yesterdayís digestions we ran a gel GEL ELECTROPHORESIS PROTOCOL . (15)
ïWe made liquid cultures of AraC+ O+AmyE 3í 12, 15, 21 LIQUID CULTURE.
ïWe ligated AraC PCR E,S dephosphated with + O+AmyE 3í LIGATION PROTOCOL.
ïWe digested 1 + O+AmyE 3í E,P LIQUID CULTURE.
We did a PCR using hot star and without MgCl2. HOT START PCR PROTOCOL
We left an RFP digestion and took the DNA from the RFP PCR which looked good. PCR PROTOCOL
From these PCR products we left the PCR digestion with EcoRI and SpeI and used enzymes from the same company. DIGESTION PROTOCOL
From the three RFP digestions we left yesterday we ran a gel to prove that our DNA wasnít being degraded, as it had been happening.
> Our DNA wasnít degraded, we will use these digestions to do the ligation.
>We did lysis of the terminator cultures by miniprep. LYSIS PROTOCOL
ïWe ran We digested 1 + O+AmyE 3í E,P with Miguelís enzymes and with Abielís enzymes LIQUID CULTURE.
ïWe transformed with ligations AraC+ O+AmyE 3í E and + O+AmyE 3í E TRANSFORMATION PROTOCOL.
ïExtracted plasmids from AraC+ O+AmyE 3í 12,15,21 [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïDigested E0040 X,S 2,3,4,5,6; ligations AraC+ O+AmyE 3í E 12,15,21; ligations AraC+ O+AmyE 3í P LIQUID CULTURE.
ïRan gel with yesterdayís digestions GEL ELECTROPHORESIS PROTOCOL . (16)
ïTransformed GusA+B0014 in two tubes TRANSFORMATION PROTOCOL.
ïMade liquid cultures from AraC+ O+AmyE 3ís transformation LB Km30 Sp 100 and control LB Km 30 Sp100 LIQUID CULTURE.
We observed two colonies, we put them in liquid LB.
We made a lysis of the ligation using miniprep.
>We did the following digestions
The results indicate that the vector only ligated with itself.
We put B0014 and E1010 digestions. DIGESTION PROTOCOL
From 24 GusA+B0014 tubes (-) we didnít do anything.
From 4 AraC+ O+AmyE 3í we extracted plasmid [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
Digested from the 4 AraC+ O+AmyE 3í E,X; X; P and GusA PCR with X,P LIQUID CULTURE.
Digested B0014 E with X B0014 X with E
ïDigested AraC+ O+AmyE 3í 1,2,3,4 with E,P LIQUID CULTURE.
We dephosphated the B0014-E/X digestion. DEPHOSPHORYLATION PROTOCOL
>We inactivated every digestion 10 minutes at 70∫C. We ran an agarose gel at 100 volts.
We put 4 ligations: LIGATION PROTOCOL
B0014 dephosphated + E1010
B0014 + E1010
At 9:30 we still didnít observe colonies of the ligation transformation.
At 1:35 20 colonies were cultivated so we could digest them (XbaI/PstI) DIGESTION PROTOCOL.
ïRan gel of digested AraC+ O+AmyE 3í 1,2,3,4 with E,P LIQUID CULTURE.
ïJoined B0014 E with X B0014 X with E digestions.
ïMade liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid LIQUID CULTURE [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL]. Transformed TRANSFORMATION PROTOCOL.
ïFrom LasR DH5a make liquid cultures and plate LIQUID CULTURE.
ïDigested O+AmyE 3í with X,P; E,S; C0080 X,P; E,S; S,P LIQUID CULTURE.
ïLigated Gus PCR X,P with B0079 S,P dephosphorylated LIGATION PROTOCOL.
ïFrom C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5a C0179 LB Cb 100 (two cultures) and a control without bacteria LIQUID CULTURE.
ïWe extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguelís transformation), Puc, B.S. control and PSB4AE [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïDue to problems with the way we did the transformations of ligations we repeated them:
GusAPCR X,P+ B0079 S,P dephosphorylated
GusAPCR X,P+ B0079 S,P dephosphorylated (-)
GusAPCR X,P+ A3 S,P dephosphorylated
GusAPCR X,P+ A3 S,P dephosphorylated (-)
ïDid the following digestions LIQUID CULTURE.
ïAraC+ O+AmyE 3í S,P 1,2,3,4
ïAraC+ O S,P
In the previous gel we observed that cultures grown were actually re-ligated vectors.
We need to put ligations again.
>E1010 doesnít look as it should, weíll extract from band. GEL EXTRACTION PROTOCOL
We ligated B0014+E1010 LIGATION PROTOCOL.
Ligated AraC+ O S,P dephosphorylated + K143002 X,P LIGATION PROTOCOL.
ïTransformed with ligations:
ïAraC+ O dephosphprylated +K143002 X,P
ïGusAPCR X,P+ A3 S,P dephosphorylated
ïGusAPCR X,P+ B0079 S,P dephosphorylated
ïTransformed the following sythesis:
ï91996 Pveg 140 bp
ï91997 ArsR-CzrA_promoter 1 194 bp
ï91998 ArsR-CzrA_promoter 2 221 bp
ï91999 ArsR-CzrA_promoter 3 213 bp
ï92000 pBad-pXyl 387 bp
ï92001 XylR 1117pb
ï92002 CI_pro_(NAND_INHIBITOR) 774
ïThese synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked. TRANSFORMATION PROTOCOL.
ïMake liquid cultures of the following transformations for tomorrow LIQUID CULTURE:
>We transformed the ligations.
The colonies from the transformation grew well, though we believe the dephosphatase isnít working properly.
We took 103 colonies and put 18 cultures to do the alkaline lysis.
Extracted plasmids from yesterdayís cultures and from K143002 cultures that were in the other lab [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P LIQUID CULTURE.
We digested with XbaI and PstI colonies 3,5,8,9,14.
ïExtracted plasmids from liquid cultures[PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL]:
PCR GusA, GusA I, PCR GusA P, PCR GusA I
ïAdd 10 µl of each primer (LW and UP).
ïAdd 3 µl of plasmid (P).
ïAdd 30.4 µl buffer.
ïAdd 5 µl Mg.
ïAdd 8 µl DNTpís.
ïAdd 42.6µl H2O miliQ.
ïAdd 1 µl RTTG polymerase.
ïCentrifuge (spin) 8 secs.
ïAdd mineral oil till the eppendorf is full.
ïPlace eppendorf 1 mL in thermocycler.
ïRun PCR with program ìBERNAî.
ï Ran a gel with: (18)
ï O+K143002 E,X; A3+GusA E,S; GusAPCR X,P
ïExtracted GusAPCR X,P digestion LIQUID CULTURE.
ïLeft the following ligation: AraC+ O+K143002 LIGATION PROTOCOL.
ïMade liquid cultures of O+K143002 (2 cultures+1 control) and AraC+ O+K143002 (2 cultures+1 control) LIQUID CULTURE.
ïLigated GusAPCR X,P+B0079 S,P dephosphorylated LIGATION PROTOCOL.
ïRan a Gel with: (19)
We ligated CI+P4 and plated in Cm25. LIGATION PROTOCOL
BBa_B0040 6I psB1A2 Amp+ plate 1
Digested 14 tubes of AraC+ O+AmyE 3í E,P LIQUID CULTURE. (19.1)
ïFrom yesterdayís PCRís :
ï E0040 plasmid 1
E0040 plasmid 2
E0040 digested 1
E0040 digested 2
We purified with PCR kit
ïRan a gel GEL ELECTROPHORESIS PROTOCOL .
ïMade 14 PCRís from AraC+ O+AmyE 3í.
ïFrom B0079+GusA ligation and B0040 transformation: ïGrew colonies.
ïMade liquid cultures LIQUID CULTURE.
ïStreaked these in a new plate.
ïDiluted plasmid E0080 2 1/50.
>We transformed the CI+P4 ligation in DH5a and plated it in Cm25. TRANSFORMATION PROTOCOL¨.
>We purified LasR PCRs so we could have linear LasR.
We digested LasR* with EcoRI and SpeI. DIGESTION PROTOCOL
We ligated CI+LasR.
We plated CI+LasR ligation in Cm25.
>We only obtained colonies in the CI+LasR plates and not in the CI.
> We put cultures so we could then do the P4+CI and CI+LasR lysis.
ïAfter running a gel with 14 PCRís AraC+ O+AmyE 3í and it did not come out as expected, we ran a gel with the ligation AraC+ O+AmyE 3í to select candidate (that weight the same as the ligation) GEL ELECTROPHORESIS PROTOCOL : (20)
ïExtracted pasmids form liquid cultures: B0049, B0079+GusA [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïAraC+ O+AmyE 3í undigested didnít run in the gel as expected, it ran similarly to AraC+ O
ï We repeated the ligation AraC+ O S,P dephosphorylated+K143002 X,P LIGATION PROTOCOL.
ï GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.
ï We ran a gel to extract.
We did lysis of the rest of the colonies for the P4CI and LasRCI ligation.
>From these lysis we left digestions with EcoRI and PstI. DIGESTION PROTOCOL
We put digestions for these lysis with E/P. DIGESTION PROTOCOL
ïRan a gel with yesterdayís digestions [GEL ELECTROPHORESIS (22)PROTOCOL]:
ArSR-CzrA 97 with S,P
ArSR-CzrA 98 with S,P
ArSR-CzrA 99 with S,P
E0040 PCR with E,S
ïLigated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P LIGATION PROTOCOL.
ïJoined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated [DEPHOSPHORYLATION PROTOCOL].
ïExtracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
ïDigested O+AmyE 3í with E,X and + O+AmyE 3í II with E,X (obtained these two from glycerols stored at -80∫C LIQUID CULTURE.
>We decided to use BcuI instead of SpeI because SpeI is not working properly.
ïWe did 2 PCRís for A3 [PCR PROTOCOL].
ïDigested K143001+PBad, pXyl with E,S
LIQUID CULTURE. (22.1)
GEL ELECTROPHORESIS PROTOCOL .
ïDid band extraction of:
1. K143001+PBad, pXyl E,S 10
2.K143001+PBad, pXyl E,S 11
3.K143001+PBad, pXyl E,S 12
ïDid 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ O+AmyE 3í 6,8,11,12 Km+ and Spectinomycin+ GLYCEROL PROTOCOL .
Pveg+XylR Chloramphenicol+ TRANSFORMATION PROTOCOL..
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ O+AmyE 3í 6 and 8 we streaked 2 plates for glycerol of each ligation.
Repeated ligation B0079+GusA.
Ligated E0040 PCR E,S+B0014 E,X dephospohylated LIGATION PROTOCOL.
Made 2 new plates from Pveg+XylR ligation and liquid cultures LIQUID CULTURE.
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight [PLASMID EXTRACTION (ìsoftî lysis) PROTOCOL].
Ligated B0079+GusA and B0040+B0014 LIGATION PROTOCOL.
We removed the plates from 37∫C.
We plated the colonies from the ligation P4CI+E1010+B0014 in Km30. We put liquid cultures of LB Km30 so we can do the lysis.
We did alkaline lysis LYSIS PROTOCOL of P4CI+E1010B0014.
>We did digestions of the lysis with EcoRI and PstI DIGESTION PROTOCOL.
We ligated LasRCI at 20 µl. LIGATION PROTOCOL.
Made liquid cultures from transformations tht were left overnight -R0079+GusA
Digested Pveg+XylR 2,3,4 with E,S.
Digested O+AmyE 3í with S,P DIGESTION.
Ran a PCR with A3 PCR (2 .6 ml tubes) [PCR PROTOCOL].
Ran a gel with
GEL ELECTROPHORESIS PROTOCOL : (23.01)
We transformed the LasRCI ligation previously plated on Cm25. TRANSFORMATION PROTOCOL
We digested in 20µl with EcoRI/PstI AmyEí5 promoter lysis. DIGESTION PROTOCOL
We counted and took LasRCI colonies and re-plated them.
ïRan a gel GEL ELECTROPHORESIS PROTOCOL : (24)
ïDigested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S LIQUID CULTURE.
ïRan a gel GEL ELECTROPHORESIS PROTOCOL : (25)
ïDephosphorylated [DEPHOSPHORYLATION PROTOCOL]:
O+AmyE 3í S,P
O+AmyE 3í II S,P
6 AraC+ O+AmyE 3í E,X
8 AraC+ O+AmyE 3í E,X
11 AraC+ O+AmyE 3í E,X
12 AraC+ O+AmyE 3í E,X
ïRan a gel with A3 PCR GEL ELECTROPHORESIS PROTOCOL : (26)
ïIt was the second time we didnít obtain a band from A3ís PCR.
ïProbably because it had been missing the buffer 2ís ethanol (ìblue lidî) from the PCR purification.
ïRan a gel woth E0040+B0014 X,P 4 to extract [GEL (27) ELECTROPHORESIS PROTOCOL]
Gen extraction protocol.
ïDid an A3 PCR [PCR PROTOCOL].
We digested the promoters with S/P to ligate it with GFP+terminator so we could characterize them. DIGESTION PROTOCOL
We left cultures with LasRCI to make lyses.