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From 2013.igem.org

Contents 1 CPEC Conditions Assay (Ralf) 2 PPTase test (Ralf) 2.1 colony PCR screening #1 2.2 MiniPrep of pRB4-10 2.3 MiniPrep PCR screening #2 2.4 Sequencing of pRB3-10 2.5 Glycerols stocks pRB3-10

CPEC Conditions Assay (Ralf)

We performed CPEC assembly of pRB13 with eight different CPEC conditions to see whether we get difeerent results.

Table 11.1 CPEC mix for assembly of pRB13

Annealing Temp [°C]	Cycles
	5	10	15
53		2: 10 ul; 4 ul backbone; 1 ul CCDB
55	3: 10 ul; 4 ul backbone; 1 ul CCDB 7: 20 ul; 8.5 ul backbone; 1.5 ul CCDB	4: 10 ul; 4 ul backbone; 1 ul CCDB 6: 20 ul; 8.5 ul backbone; 1.5 ul CCDB 8: 10 ul; 4.5 ul backbone; 0.5 ul CCDB	5: 10 ul; 4 ul backbone; 1 ul CCDB
57		1: 10 ul; 4 ul backbone; 1 ul CCDB

Table 11.2 CPEC Assembly of pRB13

BioRad T100
cycles	temp [°C]	time [s]
1	98	10
5/10/15	98	1
	53/55/57	5
	72	120
1	72	300
1	12	-

100 ul of competent TOP10 were transformed with 5 ul of reaction product and incubated for 30 hours at 37 °C.

Apparently there is no big difference using various CPEC conditions. We will continue with the standard protocol.

PPTase test (Ralf)

pRB3-10 are plasmids containing indC (pRB-3-6) or bpsA(pMM64) (pRB7-10) and a PPTase (sfp, svp, svp(pMM65), entD).

This week the blue colonies are screened and sequenced for validation.

colony PCR screening #1

Two blue colonies are picked from each plate and screened with the primers of the PPTase they should have and the

ones which they could have if something went wrong. PPTase PCR products werde used as a positive control and TOP10

without any plasmid as a negative control. So numbers 5 to 18 are subdivided into alpha and beta.

	PPTase to be screened
template	sfp (RB35/36)	svp (RB29/30)	svp(pMM65) (RB25/26)	entD (RB33/34)
	1
		2
			3
				4
	5
		6
			7
	13	14	15	8
	9
		10
			11
	16	17	18	12
	19	20	21	22

BioRad T100
cycles	temp [°C]	time [s]
1	120
30	95	60
	65	30
	72	60
1	72	300
1	12	-

Loading Scheme
5a	5b	9a	9b	1	19	2-log	6a	6b	10a	10b	2	20
7a	7b	11a	11b	3	21	2-log	8a	8b	12a	12b	4	22

The gel is not conclusive. Therefore I will repeat the screening but screen every colony for all PPTases.

MiniPrep of pRB4-10

5 ml liquid cultures of TOP10+pRB4-10 have been miniprepped using QIAquick miniprep kit. Only blue cultures have

been prepped (except pRB9 because there was no blue culture). pRB4-6 and pRB10 were prepped in duplicates (liquid

cultures of two different clones) whereas there are only single preps of pRB7-9. This is because pRB3-6 contain

indc ahich we will use for further studies. pRB6 and 10 contain entD, which is the E. coli endogenous PPTase and

was previously declared to be "inefficient" in activating bpsA [Takahashi 2007].

Concentrations of the prepped plasmids have been measured using ??ThermoScientific NanoDrop

plasmid	version	concentration [ng/ ul]
pRB4	alpha	289.7
	beta	278.7
pRB5	alpha	228.3
	beta	348.9
pRB6	alpha	428.0
	beta	432.2
pRB7	alpha	187.3
pRB8	alpha	768.2
pRB9	alpha	142.5
pRB10	alpha	112.0
	beta	212.0
water after measurement		0.7

Minipreps were reduced to a concentration of about 50 ng/ ul for sequencing and 1 ng/ ul for further studies.

MiniPrep PCR screening #2

	PPTase to be screened
template	sfp (RB35/36)	svp (RB29/30)	svp(pMM65) (RB25/26)	entD (RB33/34)
TOP10+pRB4	5	6	7	8
TOP10+pRB5	9	10	11	12
TOP10+pRB6	13	14	15	16
TOP10+pRB7	17	18	19	20
TOP10+pRB8	21	22	23	24
TOP10+pRB9	25	26	27	28
TOP10+pRB10	29	30	31	32
sfp	33
svp		34
svp(pMM65)			35
entD				36
TOP10	37	38	39	40

BioRad T100
cycles	temp [°C]	time [s]
1	95	120
30	95	60
	60	30
	72	60
1	72	300
1	12	-

Loading Scheme
5a	5b	9a	9b	13a	13b	17	21	25	29a	29b	sfp	2-log	6a	6b	10a	10b	14a	14b	18	22	26	30a	30b	svp
7a	7b	11a	11b	15a	15b	19	23	27	31a	31b	svp(pMM65)	2-log	8a	8b	12a	12b	16a	16b	20	24	28	32a	32b	entD

pRB9 is negative. All screens are positive in the svp(pMM65) and entD section. In entD this could be due to genomic

DNA in the miniprep. svp(pMM65) is also positive in all plasmids, which is strange. Maybe the PCR conditions have

been too tolerant. Nevertheless the bands of the plasmids which should carry the respective PPTase appear to be

stronger. We will sequence the plasmids anyways.

Sequencing of pRB3-10

plasmid minipreps have been sequenced by GATC [reference] using the sequencing primers VF2 and VR. The sequencing

results show that all the inserts are correct except for pRB9, which isn't a pity because we have previously shown

with pKH1-2 that the combination of bpsA(pMM64) and svp(pMM65) successful produces indigoidine.

plasmid	Primer	ind synthetase/ PPTase	sequencing result/ reference
pRB3	VF2	indC
			seq2
	VR	sfp-rev
			seq2
pRB4	VF2	indC
			seq2
	VR	svp(pMM65)-rev
			seq2
pRB5	VF2	indC
			seq2
	VR	svp-rev
			seq2
pRB6	VF2	indC
			seq2
	VR	entD-rev
			seq2
pRB7	VF2	bpsA
			seq2
	VR	sfp-rev
			seq2
pRB8	VF2	bpsA
			seq2
	VR	svp(pMM65)-rev
			seq2
pRB9	VF2	bpsA
			seq2
	VR	svp-rev
			seq2
pRB10	VF2	bpsA
			seq2
	VR	entD-rev
			seq2

In the following pRB4-beta, pRB5-alpha, pRB5-beta, pRB6-alpha and pRB10-alpha have been selected so that there are

single versions of pRB3-10 for furhter experiments.

Glycerols stocks pRB3-10

3 stocks each were made using 2 mL liquid cultures (48 h at 37 °C 200 rpm) of TOP10+pRB3-10, respectively.