IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-10

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Oscillation model

Jeff finished adding sinusoidal and feedback alpha functions to his model. He will upload the code later.

Meeting

We had our weekly team meeting today. Several people made some good points and suggestions:

• For solution #2, we can simply wash the exogenous chemical away (e.g. lactose) to disable expression at will. Pam expressed reservations about the heat shock in solution #1. We can substitute solution #2 for solution #1 by washing. In other words, we can fulfill both experiments of interest-- constant KaiABC production and initial KaiABC production-- with just one promoter.
• As Nick pointed out in an earlier email, even if we can't see oscillation, we can still at least show that something is happening. KaiC stays unphosphorylated in the absence of KaiA and KaiB. If we notice that KaiC is, say, 50% phosphorylated, then, even if we can't observe oscillation, we know KaiABC are interacting.
• Pam suggested that we synthesize our KaiABC BB constructs in parallel with PCRing out of PCC 7942, to cover our bases in case the PCR takes a long time.
• Regarding PCR, William pointed out that we might obtain a higher yield by lysing our cells chemically instead of relying on temperature.
• Also, we should do more research on PCR protocols for cyanobacteria.
• In the future, we should organize our presentations a little better and give a high-level overview at the start.

New cultures of PCC 7942

Our new culture grew quickly

Both flasks from Friday showed growth. The flask with thiosulfate was rich green while the flask without was paler. We were surprised and pleased to find that our cyanobacteria was growing so quickly.

DNA Synthesis companies

1. Blue Heron Biotechnology, Inc.
   • Advertised as 2-4 weeks, but teams have had problems getting sequences on time in the past.
2. Codon Devices, Inc.
3. DNA 2.0, Inc.

Add more links here. Additional information would be useful.

Frozen stocks

Peng made frozen stocks of our newly grown PCC 7942. (Peng fill this in)

Mega PCR Debug

LABEL	SUBSTRATE	PRIMERS	TAQ + ATP	[Mg+]	NTP	ANNEALING TEMP	PCR MACHINE	REACTION	PWNED BY
1	Cy7942	Cy primers	Old Taq	-	Old NTPs	65\'	1	D	Peng
2	Cy7942	Cy primers	New Taq	2mM	Old NTPs	65\'	1	A	Hetmann
3	Cy7942	Cy primers	Old Taq	-	New NTPs	65\'	1	D	Peng
4	Cy7942	Cy primers	New Taq	2mM	New NTPs	65\'	1	A	Hetmann
5	Cy7942	Cy primers	New Taq	4mM	New NTPs	65\'	1	B	Dave
6	Cy7942	Cy primers	New Taq	6mM	New NTPs	65\'	1	C	Jeff
7	Biobrick Miniprep	Biobrick Primer	Old Taq	-	Old NTPs	65\'	1	E	Peng
8	Biobrick Miniprep	Biobrick Primer	New Taq	2mM	Old NTPs	65\'	1	A	Hetmann
9	Biobrick Miniprep	Biobrick Primer	Old Taq	-	New NTPs	65\'	1	E	Peng
10	Biobrick Miniprep	Biobrick Primer	New Taq	2mM	New NTPs	65\'	1	A	Hetmann
11	Biobrick Miniprep	Biobrick Primer	New Taq	4mM	New NTPs	65\'	1	B	Dave
12	Biobrick Miniprep	Biobrick Primer	New Taq	6mM	New NTPs	65\'	1	C	Jeff
13	Cy7942	Cy primers	New Taq	2mM	New NTPs	55\'	2	A	Hetmann
14	Cy7942	Cy primers	New Taq	4mM	New NTPs	55\'	2	B	Dave
15	Cy7942	Cy primers	New Taq	6mM	New NTPs	55\'	2	C	Jeff
16	Biobrick Miniprep	Biobrick Primer	New Taq	2mM	New NTPs	55\'	2	A	Hetmann
17	Cy7942	Cy primers	New Taq	2mM	New NTPs	65\'	1	A	Hetmann
18	Cy7942	Cy primers	New Taq	4mM	New NTPs	65\'	1	B	Dave
19	Cy7942	Cy primers	New Taq	6mM	New NTPs	65\'	1	C	Jeff
20	Biobrick Miniprep	Biobrick Primer	New Taq	2mM	New NTPs	65\'	1	A	Hetmann
21	Cy7942	Cy primers	New Taq	2mM	New NTPs	55\'	2	A	Hetmann
22	Cy7942	Cy primers	New Taq	4mM	New NTPs	55\'	2	B	Dave
23	Cy7942	Cy primers	New Taq	6mM	New NTPs	55\'	2	C	Jeff
24	Biobrick Miniprep	Biobrick Primer	New Taq	2mM	New NTPs	55\'	2	A	Hetmann

PCR Reactions

REACTION A	REACTION B	REACTION C	REACTION D	REACTION E
2 mM [Mg]	4 mM [Mg]	6 mM [Mg]
10 μL template	10 μL template	10 μL template	5uL template	1 uL template
10 μL 10x buffer	10 μL 10x buffer	10 μL 10x buffer	5 uL 10x buffer	5 uL 10x buffer
2 μL 10 mM dNTP	2 μL 10 mM dNTP	2 μL 10 mM dNTP	1 uL 10mM dNTP
2 μL Primer 1	2 μL Primer 1	2 μL Primer 1	1 uL Primer 1	1 uL Primer 2
2 μL Primer 2	2 μL Primer 2	2 μL Primer 2	1 uL Primer 2	1 uL Primer 2
1 μL Taq	1 μL Taq	1 μL Taq	0.5 uL Taq	0.5 uL Taq
73 μL dH20	71 μL dH20	69 μL dH20	37 μL dH20	41 μL dH20
-	2 μL 100 mM Mg	4 μL 100 mM Mg

Notes

1) Some were made with 10uL positive control instead of 2uL; may affect results (Jeff and Dave's samples)

2) The Run tims is:

PCR5: @ 55C anneal:

 #94C, 15'
 #94C, 30"
 #56C, 30"
 #72C, 3.5'
 #Cycle to step 2, 7x
 #94C, 30"
 #55C, 30"
 #72C, 3.5'
 #Cycle to step 6, 30x
 #72C, 5'
 #4C hold

PCR6: @ 65C anneal:

 #94C, 15'
 #94C, 30"
 #56C, 30"
 #72C, 3.5'
 #Cycle to step 2, 7x
 #94C, 30"
 #65C, 30"
 #72C, 3.5'
 #Cycle to step 6, 30x
 #72C, 5'
 #4C hold