Team:Heidelberg/Model test2

iGEM TEAM HEIDELBERG 2017

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COLLAPSE NAVIGATION

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Title

Subtitle

Successful in vivo directed evolution by PREDCEL and PACE requires the thorough consideration of experimental parameters, e.g. phage propagation times, culture dilution rates and inducer/inhibitor concentrations. We employed extensive ODE-based and stochastic modeling to identify the most sensitive parameters and adapt our experiments accordingly. First, we calibrated our models using phage propagation experiments from our wet lab complemented with literature data. Simulations showed that the phage titer is highly sensitive to culture dilution rates. We simulated batch times and transfer volumes for PREDCEL and corresponding flow rates for PACE to determine optimized conditions for gene pool selection while avoiding phage washout. We also estimated phage titer monitoring intervals for cost/labor efficient QC as well as inducer/inhibitor concentrations required to express the required mutagenic polymerases. Finally, provide a web-based, fully interactive modeling platform, not only extensively employed by our wet lab, but highly informs future iGEM teams building on our work.

Table X: Title Subtitle

TIme	Date	Operator	Sample	Action	DONE ToDo:	OD Turbidostat 2	Stock ID	TO DOs:
12:00	24/10/2017	MP	-	Started filling the turbidostat (T2) with medium			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
13:30	24/10/2017	MP	-	Inoculation of turbidostat 2 with 50 ml Stock culture			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
14:00	24/10/2017	MP	-	Measured OD		"0	18"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
15:00	24/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
16:00	24/10/2017	MP	-	Measured OD		"0	511"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
17:00	24/10/2017	MP		"Measured OD	started filling the lagoon and the arabinose pump"		"0	548"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
18:00	24/10/2017	MP	1	Inoculation with 1 ml phage supernatant		"0	514"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
19:00	24/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
20:00	24/10/2017	MS	1	"Measured OD	Took samples L1	T1 - 1"		"0	637"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
21:00	24/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
22:00	24/10/2017	MP		"Measured OD	Flow rate for lagoon was to high from previous filling ( 7 ml/min in contrast to 1	666 ml/min) - potential washout "		"0	791"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
23:00	24/10/2017	MP		"Lagoon was infected with 0	8 ml 10^8 PFU/ml phages to counteract the potential washout "		"0	818"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
00:00	25/10/2017	MP	2	"Measured OD	Took samples L1	T1 - 2"		"0	833"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
01:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
02:00	25/10/2017	MS		Measured OD		"0	789"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
03:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
04:00	25/10/2017	MS	3	"Measured OD	Took samples L1	T1 - 3"			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
05:00	25/10/2017					"0	792"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
06:00	25/10/2017	MS		Measured OD			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
07:00	25/10/2017					"0	817"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
08:00	25/10/2017	TB	4	"Measured OD	Took samples L1	T1 - 4"	Took samples		199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
09:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
10:00	25/10/2017	TB		Measured OD		"0	816"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
11:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
12:00	25/10/2017	TB		Measured OD	Done PCR	"0	81"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
13:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
14:00	25/10/2017	PP		Measured OD		"0	843"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
15:00	25/10/2017				inoculated growth for plaque assay		199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
16:00	25/10/2017	PP	5	"Measured OD	Took samples L1	T1 - 5"		"0	833"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
17:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
18:00	25/10/2017	PP		Measured OD		"0	836"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
19:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
20:00	25/10/2017	MP		Measured OD		"0	862"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Plaque Assay
21:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
22:00	25/10/2017	MP		Measured OD		"0	81"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
23:00	25/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
00:00	26/10/2017	LP	6	"Measured OD	Took samples L1	T1 - 6"	Done PCR	"0	809"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
01:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
02:00	26/10/2017	CG		Measured OD		"0	845"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
03:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
04:00	26/10/2017	CG		Measured OD		"0	864"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
05:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
06:00	26/10/2017	CG		Measured OD		"0	836"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
07:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
08:00	26/10/2017	TB	7	"Measured OD	Took samples L1	T1 - 7"		"0	831"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
09:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
10:00	26/10/2017	TB		Measured OD		"0	827"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
11:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
12:00	26/10/2017	TB		Measured OD			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
13:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
14:00	26/10/2017	TB		Measured OD		"0	738"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
15:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
16:00	26/10/2017	TB	8	"Measured OD	Took samples L1	T1 - 8"	"Took samples L1	T1"	"0	652"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
17:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
18:00	26/10/2017			Measured OD			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
19:00	26/10/2017	MS				"0	582"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
20:00	26/10/2017			Measured OD	Plaque assay was performed successfully		199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Plaque Assay
21:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
22:00	26/10/2017	MS		Measured OD		"0	633"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
23:00	26/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
00:00	27/10/2017	PP	9	"Measured OD	Took samples L1	T1 - 9"		"0	511"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
01:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
02:00	27/10/2017	MS		Measured OD		"0	692"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
03:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
04:00	27/10/2017	MS		Measured OD		"0	937"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
05:00	27/10/2017					"0	945"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
06:00	27/10/2017	MP		Measured OD		"could not be measured	as lagoon medium level was too high and therefore pump where set off for a short time "	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
07:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
08:00	27/10/2017	MS	10	"Measured OD	Took samples L1	T1 - 10"	"Phage detection PCR with samples 8	9 and 10 for Dickinson-Phage and PI-Phage"	"0	912"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
09:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
10:00	27/10/2017	PP		Measured OD		"0	903"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
11:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
12:00	27/10/2017	LP		Measured OD	see above	"0	924"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
13:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
14:00	27/10/2017	MP		Measured OD		"0	942"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
15:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
16:00	27/10/2017	PP	11	"Measured OD	Took samples L1	T1 - 11"	took samples	"0	942"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
17:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
18:00	27/10/2017	MP		Measured OD		"0	822"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
19:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
20:00	27/10/2017	MK		Measured OD		"0	59"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Plaque Assay
21:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
22:00	27/10/2017	CG		Measured OD		"0	61"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
23:00	27/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
00:00	28/10/2017	MP	12	"Measured OD	Took samples L1	T1 - 12"	Phage detection PCR with sample 11	"0	664"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
01:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
02:00	28/10/2017	CG		Measured OD		"0	809"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
03:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
04:00	28/10/2017	MP		Measured OD	PCR: negative - phage washout	"0	820"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
05:00	28/10/2017			"Flow rate was adjusted: 1 ml/min	lagoon was infected by pooled samples 6	7 and 8 "			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
06:00	28/10/2017	MP		Measured OD			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
07:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
08:00	28/10/2017	MP	13	"Measured OD	Took samples L1	T1 - 13"		"0	667"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
09:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
10:00	28/10/2017	PP		Measured OD		"0	62"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
11:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
12:00	28/10/2017	PP		Measured OD	Phage PCR was performed	"0	768"	199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)	TO DO: Phage-PCR
13:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
14:00	28/10/2017	PP		Measured OD			199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
15:00	28/10/2017						199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4)
16:00	28/10/2017	PP	14	"Measured OD	Took samples L1	T1 - 14"	FINAL PROBES

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