Glasgow/Wetlab/Week7

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Week 7

Monday 13th August 2007

1. Lynsey did colony PCR (see Protocol 9) on the following E. coli TOP10 transformants to check that their plasmids carry our expected inserts:
   
   

   Plasmid	Expected Insert	Primers
   pCR4	Pu	Pu_Prefix_After & Pu_Suffix_After
   pCR4	Pu	Pu_Prefix_Emma & Pu_Suffix_Emma
   pCR4	Pr	Pr_Prefix_1 & Pr_Suffix_1
   pCR4	XylR	XylR_Prefix_1 & XylR_Suffix_1
   pCR4	XylR & Pr	Pr_Prefix_1 & XylR_Suffix_1

   
   Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.

2. Maija did colony PCR (see Protocol 9) on the following E. coli DB3.1 transformants to check that their plasmids carry our expected inserts:
   
   

   Plasmid	Expected Insert	Primers
   pSB3K5 (4/6B)	(*m*) B1 17	(*m*)_for_1 & BBs_Methyl_2
   pSB1AC3 (3/20G)	(*m*) B1 17	(*m*)_for_1 & BBs_Methyl_2
   pSB3K5 (4/6B)	(*m*) C5 24	(*m*)_for_1 & (*m*)_rev_1
   pSB1AC3 (3/20G)	(*m*)	(*m*)_for_1 & (*m*)_rev_1
   pSB3K5 (4/6B)	DntR	DntR_Prefix_1 & DntR_Suffix_2
   pSB1AC3 (3/20G)	DntR	DntR_Prefix_1 & DntR_Suffix_2

   
   

   Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.

3. Maia took the following BioBricks from the kit plates and transformed (see Protocol 2) into E. coli TOP10 cells:
   
   

   BioBrick Part	Function	Chassis	Plasmid	Kit Plate Location (Plate/Well)
   Bba_B0014	Double terminator	E. coli TOP10	pSB1AK3	1/1G
   Bba_B0015	Double terminator	E. coli TOP10	pSB1AK3	1/1I
   "	"	"	"	3/3O
   Bba_J61100	RBS	E. coli TOP10	pSB1A2	4/12N
   Bba_J61101	RBS	E. coli TOP10	pSB1A2	4/12J
   Bba_J61102	RBS	E. coli TOP10	pSB1A2	4/12L
   Bba_J61103	RBS	E. coli TOP10	pSB1A2	4/12P
   Bba_E0430	EYFP + RBS + terminator	E. coli TOP10	pSB1A2	1/11A
   Bba_E0422	ECFP + RBS + terminator + LVA tag	E. coli TOP10	pSB1AK3	1/11G
   Bba_E0432	EYFP + RBS + terminator + LVA tag	E. coli TOP10	pSB1A2	4/11C

4. To ensure successful cloning of the PCR products (*a→g*), (*a→d*) and (*d→g*) from 10/07, 1ul of GoTaq was added to each 50ul PCR reaction and extended for 10 min at 72°C. Four large lanes of (*a→g*) were run and each had a band of interest of ~8kb . One lane of each (*a→d*) and (*d→g*) was ran and only that of (*a→d*) had a band of interest of ~3kb. Each gel was 1% agarose, 50v for 40 min).
5. (*a→g*) and (*a→d*) were gel extracted (see Protocol 11 ), cloned into TOPO vectors(see Protocol 12 ), transformed (see Protocol 13 )and grown overnight at 37°C, each reaction divided into 100ul and ~200ul per plate.

Tuesday 14th August 2007

1. Each plate of transformations of (*a→g*) and (*a→d*) grew several hundred colonies. Eight colonies from plates spread with 100ul of reaction were selected for colony PCR using Reddymix and Touch 2 program (see Protocol 9). Each selected colony was used in a reaction with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1. No expected bands were of 2kb were observed in either reaction.
2. Overnights of Colonies 1 and 2 from each of the above plates (spread with 100ul of transformants) were inoculated overnight in carb LB.
3. Lynsey took her overnight cultures of P_u and P_r and
   • miniprepped them (see Protocol 5),
   • digested out their inserts(see Protocol 7), then
   • ligated (see Protocol 15) them into pSB3K5.
4. Maia did colony PCR (see Protocol 9; program Touch 2) using primers VF2 and VR on the BioBricks we transformed from the kit yesterday, confirming on the basis of product size that our transformants carried the expected plasmids.

Wednesday 15th August 2007

1. Miniprepped overnights according to QIAGEN manual (see Protocol 5), and were labelled as follows:

   Label	1/1	1/2	2/1	2/2	3/1	3/2	4/1	4/2	5/1	5/2
   Plate	1	1	2	2	3	4	4	5	5
   Colony	1	2	1	2	1	2	1	2	1	2
   Expected Gene	(*a→g*)	(*a→g*)	(*a→g*)	(*a→g*)	(*a→g*)	(*a→g*)	(*a→g*)	(*a→g*)	(*a→d*)	(*a→d*)

2. PCR was done for each of the above minipreps with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1, used Reddymix and Touch2 (see Protocol 9). There is no evidence of a 7 gene operon insert in any of the colonies.
3. Lynsey repeated amplification of XylR and XylR+Pr with KOD polymerase (see Protocol 9) using the primers listed below and subsequently cloned into TOPO vector (see Protocol 12) and transformed TOP10 cells (see Protocol 13).
   • Primer Pairs:
     • XylR Prefix + XylR Suffix
     • Pr Prefix + XylR Suffix
     • Pr_for_2 + Xylr_rev_2
4. Maija performed PCR based site-directed mutagenesis for (*m*) to correct one PstI site and (*s*) to correct two PstI sites. Only one PstI site can be corrected at the time so Maija started working with both sites separately and proceeding to the second PstI site with the construct where either one has been corrected.

   Biobrick + Construction Vector	Primer Pair
   (*m*) + 3/20G	(*m*)_SDM_PstI_1_for + (*m*)_SDM_PstI_1_rev
   (*m*) + 4/6B
   (*s*) + 3/20G	(*s*)_SDM_PstI_1_for + (*S*)_SDM_PstI_1_rev
   (*s*) + 4/6B
   (*s*) + 3/20G	(*s*)_SDM_PstI_2_for + (*s*)_SDM_PstI_2_rev
   (*s*) + 4/6B

   KOD polymerase was used during the mutagenesis PCR, (see Protocol 9) and (seeProtocol 17)

Thursday 16th August 2007

1. Repeated digests from 9/08 with PstI and EcoRI of minipreps thought to contain (*a→g*). There is a possibility that colony 4/1 contains the (*a→g*) insert.
2. 5 ml overnight cultures done with LB kanamycin or LB carbenicillin from the transformed cell colonies 1-18 after the first round of the site-directed mutagenesis in order to get rid of PstI sites inside our becoming BioBrick genes (*m*) and (*s*).
   1. (*m*) + 4/6B (kanamycin)Site-Directed Mutagenesis
   2. (*m*) + 4/6B (kan) SDM
   3. (*m*) + 4/6B (kan) SDM
   4. (*m*) + 3/20G (carbenicillin) SDM
   5. (*m*) + 3/20G (carb) SDM
   6. (*m*) + 3/20G (carb) SDM
   7. (*s*) + 4/6B (kan) SDM 1. (The first PstI site)
   8. (*s*) + 4/6B (kan) SDM 1.
   9. (*s*) + 4/6B (kan) SDM 1.
   10. (*s*) + 3/20G (carb) SDM 2. (The second PstI site)
   11. (*s*) + 3/20G (carb) SDM 2.
   12. (*s*) + 3/20G (carb) SDM 2.
   13. (*s*) + 4/6B (kan) SDM 2.
   14. (*s*) + 4/6B (kan) SDM 2.
   15. (*s*) + 4/6B (kan) SDM 2.
   16. (*s*) + 3/20G (carb) SDM 1.
   17. (*s*) + 3/20G (carb) SDM 1.
   18. (*s*) + 3/20G (carb) SDM 1.

Friday 17th August 2007

1. Christine did more PCR of the minipreps of the colonies suspected of containing the 7 gene operon using Reddy mix, Touch 2 and the pimer combinations below for each miniprepped colony (see Protocol 9). The result gave no evidence of the insert being present.
   • *M13_For and *B_EcoRI_SDM_RevI
   • *M13_Rev and *B_EcoRI_SDM_RevI
2. Maija miniprepped the mutagenised cell cultures 1-18 and performed a PstI restriction digest with Roche's PstI restriction enzyme to verify whether the mutagenesis worked or not.
3. On the same 1% agarose gel Maija run the original plasmids which had not been mutagenised but had been digested with the same Roche's PstI restriction enzyme as the mutagenised ones. The original plasmids were the following:
   • (*m*) + 4/6B
   • (*m*) + 3/20G
   • (*s*) + 4/6B
   • (*s*) + 3/20G
4. From the gel it seems that the (*m*) minipreps 1., 2., 4. and 5. were successful and thus no PstI site could be seen anymore.
5. The ten (*s*) minipreps 7.-16. were successful and thus only one PstI site was detected. Maija is going to continue with mutagenising the other PstI site next week.