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From 2013.igem.org

Contents 1 Preparation for T-Domain exchange 1.1 Assembly of pRB19 1.2 Assembly of pRB15-18 1.3 Assembly of pKH4

Preparation for T-Domain exchange

Assembly of pRB19

PCR indC(ccdB) RB27/28 and pSB1C3 RB21/22 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 14.x PCR indC(ccdB) for pRB19

RB27/46 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
5	98	1
	55	5
	72	60
30	98	1
	72	60
1	72	180
1	12	-

Table 14.x PCR indC(ccdB) for pRB19

RB27/46 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
10	98	1
	td 55	5
	72	70
25	98	1
	60	5
	72	70
1	72	180
1	12	-

Gel Extraction with QIAquick gel extraction kit.

Table 13.x CPEC Assembly Master Mix pRB19 second try

Plasmid	Fragment 1	Molarity [nM]	Volume in ul	Fragment 2	Molarity [nM]	Volume in MM	DpnI Master Mix total [ul]
pRB19	indC(ccdB) from pRB14	14.6	14	pSB1C3 RB21/22	105.3	3	20

Digest ran for 1 hour at 37 °C, was put on a agarose gel, gel extracted and eluted in 15 ul water.

Concentration was measured using NanoVue: 30 ng/ ul. 10 ul were put together with 10 ul of Phusion

Flash HF Master Mix for standard CPEC assembly. OneShot and TOP10 cellc were transformed using different amounts of CPEC product.

Ten colonies were screened using iTaq with KH9/VR and RB35/VR, respectively, to see whether the assembly worked and sfp was kicked out.

The screening was positive, so probe 10 was kept for 10 ml liquid culture (TB+Cm). Miniprep of 4 ml yielded 69.4 ng/ ul in 50 ul. Miniprep was used for PCR with primers KH3/4 to get the fragment pRB19 without ccdB for insertion of T-Domains via CPEC. First we tried two different polymerases at two different conditions each. The first is Phusion Flash

HF 2x Master Mix (ThermoScientific) and the other is Phusion HF 2x Master Mix (NEB).

Table 14.x Test PCR KH3/4 from pRB19

KH3/4 in BioRAD T100 with Phusion HF (NEB)
Cycles	Temperature [°C]	Time [s]
1	98	30
30	98	15
	57-60	15
	72	3.30
1	72	10.00
1	12	-
KH3/4 in BioRAD T100 with Phusion Flash HF (ThermoScientific)
Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	57-60	5
	72	2.00
1	72	6.00
1	12	-

Table 14.x synT-PCR

KH5/6 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	30
45	98	5
	60	15
	72	15
1	72	60
1	12	-

Gel extraction and measurement using NanvoVue. The final table of T-domain concentration is as

follows:

Table 13.x Concentrations of T- and TTE-domains to be shuffled

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
ccdB	121.2	700	262.33
indC-T	192.4	200	1457.58
bpsA-T	198.9	200	1506.18
entF-T	64.5	200	488.6
tycA1-T	184.2	200	1395.45
tycC6-T	198.6	200	1504.55
delH4-T	175.0	200	1325.76
delH5-T	162.4	200	1230.30
plu2642-T	79.0	200	598.5
plu2670-T	31.5	200	238.6
synT1	43	200	325.8
synT2	37.5	200	284.1
synT3	40.5	200	306.8
synT4	36	200	272.7
synT5	37.5	200	284.1
synT6	37	200	280.3
synT7	40.5	200	306.8
bpsA-TTE	92.9	1000	140.8
entF-TTE	-	1000	-
tycC6-TTE	21	1000	31.8
delH5-T	22.9	1000	34.7

Assembly of pRB15-18

pRB15-18 were unable to be sequenced with various primers. We now use pSB3K3 backbone from plate 3 of the 2013 spring distribution and assemble those PPTase-plasmids de novo.

Table 14.x PCR pSB3K3 with RB21/63 #1

RB21/63 in BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
14	98	1
	td 66	5
	72	60
16	98	1
	68	5
	72	60
1	72	180
1	12	-

PCR was run with with a fragment of the Tyrocidine group with slightly suboptimal conditions. It will be repeated using otimal conditions.

Table 14.x PCR pSB3K3 with RB21/63 #2

RB21/63 in BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	td 61	5
	72	50
20	98	1
	65	5
	72	50
1	72	180
1	12	-

Gel extraction yielded 144.2 ng/ ul (Nanodrop)

CPEC was performed using pSB3K3 gel extraction and gel extractions made for assembly of pRB3-10. TOP10 cells were transformed using standard protocol.

Table 14.x CPEC Assembly pRB15-18

Plasmid	Fragment 1	Molarity [nM]	Volume [ul]	Fragment 2	Molarity [nM]	Volume [ul]	CPEC Master Mix total [ul]
pRB15	pSB3K3(RB21/63)	87.4	2	sfp	713.4	1	6
pRB16	pSB3K3(RB21/63)	87.4	2	svp	90.7	4	12
pRB17	pSB3K3(RB21/63)	87.4	2	entD	674.1	1	6
pRB18	pSB3K3(RB21/63)	87.4	1	delC	118.1	2	6

Cells were plated on LB+Kanamycin and incubated at 37 °C for 24 hours.

Five colonies of each plate were screened using iTaq polymerase and two primer combinations for each colony. This is VF2/PPTase_rv primer and PPTase_fw/VR primer.

pRB16 is obviously wrong. We checked the PCR product we used for the assembly and recognized that it's wrong, so we have to amplify it again from S. verticillus. DelC screenings show another small band, we have too check whether the primers bind elsewhere on the plasmid and run a negative control with those primers.

Assembly of pKH4

pKH4 is a pRB3 derived version of an indigoidine synthetase expression plasmid w/o sfp as activating PPtases. So the two plasmid strategy can be conducted (at first) with this plasmid instead to rely on pRB21. Basically the idea is to use restriction sites before and after sfp to discard it and to religate with another sequence to yield a functional backbone.

pRB3 has a BamHI before and a NheI cutting site after sfp. pMM64 and pMM65 both can be cut with a combination of BglII and XbaI to yield for example a 164 bp long fragment with compatible ends to the linearized pRB3. This fragment unfortunately contains a T7lac hybrid promoter which could interfere with our used primers, but still is worth a shot.

• digest mix volume: 30 µl (3 µl NEBuffer 3.1, 6.5 µl pMM65 (~481 ng), 0.3 µl (BglII, XbaI) each, 19.9 µl H2O)

Resulting gel of digestion of pMM65 with BglII and XbaI in order to gain sequence with cohesive ends to linerized pRB3 w/o sfp.

• digestion gave expected bands at 2 kbp and 2.4 kbp but band at ~160 bp is missing. Reaction was carried out with not enough DNA.
• prepared ON for pMM64/65 MP as well as pRB3 MP
• the next day ON were extended to 2x 4 ml LB cultures which were then

prepped after several hours of incubation at 37 °C

• MP with cultures: DNA conc. measurement with NanoVue gave: 280

ng/µl (pMM64), 225 ng/µl (pMM65), 250 ng/µl (pRB3); analysis gel shows different concentration for pMM65 (~15 ng/µl ?)

Analytical gel for verification of MP pMM64/65 and pRB3: concentrations measured with NanoVue seem to be ok for pMM64 and pRB3, but much less for pMM65. 2log ladder lane contain 420 ng each; 100 V, TAE

• prepare digestion (20 µl), 37 °C, 1.5 h:
  • pMM64: 2 µl NEBuffer 3.1, 17 µl pMM64, 0.5 µl (BglII, XbaI)
  • pRB3: 2 µl NEBuffer CutSmart, 10 µl pRB3, 0.5 µl (BamHI, NheI), 7

µl H2O

Digestion of pMM64 (BglII, XbaI) and pRB3 (BamHI, NheI) were desired bands for insert of ~170 bp and backbone-indC of ~6300 bp were cut out. 100 V, TAE

• after gelextraction (elution in 20 µl) ligation was conducted with 2

µl 10x T4 ligase buffer, 1 µl T4 ligase, 2 µl pRB3 Dig_GE, 8 µl pMM64 Dig_GE and 7 µl H2O for 40 min at RT

Analytical gel of 1 µl of ligation mix, compared to 0.5 µl of pRB3 digestion with (BamHI, NheI). Additionally 1 µl of MP pMM65 was put on gel to verify previous concentration measurement. 1.5 µl of log2 ladder, 100 V, TAE

• transformation in TOP10 with 1 µl and 5 µl of ligation mix