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

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Newer entries at the bottom.

Contents

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
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
1Cy7942Cy primersOld Taq-Old NTPs65\'1DPeng
2Cy7942Cy primersNew Taq2mMOld NTPs65\'1AHetmann
3Cy7942Cy primersOld Taq-New NTPs65\'1DPeng
4Cy7942Cy primersNew Taq2mMNew NTPs65\'1AHetmann
5Cy7942Cy primersNew Taq4mMNew NTPs65\'1BDave
6Cy7942Cy primersNew Taq6mMNew NTPs65\'1CJeff
        
7Biobrick MiniprepBiobrick PrimerOld Taq-Old NTPs65\'1EPeng
8Biobrick MiniprepBiobrick PrimerNew Taq2mMOld NTPs65\'1AHetmann
9Biobrick MiniprepBiobrick PrimerOld Taq-New NTPs65\'1EPeng
10Biobrick MiniprepBiobrick PrimerNew Taq2mMNew NTPs65\'1AHetmann
11Biobrick MiniprepBiobrick PrimerNew Taq4mMNew NTPs65\'1BDave
12Biobrick MiniprepBiobrick PrimerNew Taq6mMNew NTPs65\'1CJeff
        
13Cy7942Cy primersNew Taq2mMNew NTPs55\'2AHetmann
14Cy7942Cy primersNew Taq4mMNew NTPs55\'2BDave
15Cy7942Cy primersNew Taq6mMNew NTPs55\'2CJeff
16Biobrick MiniprepBiobrick PrimerNew Taq2mMNew NTPs55\'2AHetmann
        
17Cy7942Cy primersNew Taq2mMNew NTPs65\'1AHetmann
18Cy7942Cy primersNew Taq4mMNew NTPs65\'1BDave
19Cy7942Cy primersNew Taq6mMNew NTPs65\'1CJeff
20Biobrick MiniprepBiobrick PrimerNew Taq2mMNew NTPs65\'1AHetmann
        
21Cy7942Cy primersNew Taq2mMNew NTPs55\'2AHetmann
22Cy7942Cy primersNew Taq4mMNew NTPs55\'2BDave
23Cy7942Cy primersNew Taq6mMNew NTPs55\'2CJeff
24Biobrick MiniprepBiobrick PrimerNew Taq2mMNew NTPs55\'2AHetmann

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
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