IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-13

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Contents

Incubator status

  • Removed
    • ...
Click here for a map of the lower shelf(2006-07-13)
Click here for a map of the lower shelf
(2006-07-13)

Experimental Results

The plates grown from yesterday (2nd Topo&Transformation) show the following:

Positive Control: Many colonies

Negative Control: No colonies

Tranformed Plate: One in the middle, three+ on the edge of the plate.

DNA Miniprep of Topo+KaiABC plasmid

Using the regular Qiagen Protocol (Vs.2 of DNA miniprep booklet, p22) - DNA is stored in a 1.5uL blue marked microcentrifuge tube, 50uL dH20, 62.6ng/uL.

Liquid culture growth

Hetmann grew another liquid culture for use for glycerol stock.

Site-Specific Mutagenesis PCR

Refer to the image for details; this is the "crossover pcr" step.

Refer to this for PCR overview
Refer to this for PCR overview

For this step, we are going to create 4 PCR constructs with primers which will create the site mutation we want to eliminate the restriction sites.

The 4 sites are:

    1. kABC_3_9
    2. kABC_10_7
    3. kABC_8_5
    4. kABC_6_4

Where:

  • 3=crossF
  • 4=crossR
  • 5=pst1R
  • 6=pst1F
  • 7=pst2R
  • 8=pst2F
  • 9=eco1R
  • 10=eco1F

and:

  • the kABC_x_y signifies the sequence bounded by primers 'x' and 'y'

The cycle for this PCR is:

 *#95@15'
 *#94@30"
 *#56@30"
 *#72@3'30"
 *#Cycle to step 2, 40x
 *#72@5'
 *#4@forever

Each pot should have:

Using the Roche Vent PCR Kit
0.5 μL template
5 μL 10x buffer
1 μL 10 mM dNTP
1 μL Primer 1
1 μL Primer 2
0.5 μL Taq
41 μL dH20

PCR reactions

David, Hetmann, and Jeff each made 5 PCR reactions: 1 for each of the 4 mutation sites above, plus a negative control.

All 15 reactions were run in the PCR1 machine using the cycle above.

Future Experiment Planning

Experiment 1

Goal
To verify the existence of KaiA and KaiBC transcription in E. coli.
Procedure 
  1. Attach a fluorescent tag onto KaiA/B/C.
  2. Insert the tagged KaiA/B/C into E. coli.
  3. Induce the attached promoter.
  4. Measure the E. coli fluorescence.
    • The phosphorylation state of KaiC should't have an effet on its fluorescence.

Ideally, we would see some fluorescence (oscillating fluoresence would be even better), which would indicate that our promoters are working and the Kai proteins are being transcribed.

Experiment 2

Goal
To measure the interaction between KaiA and KaiBC in E.coli.
Procedure
  1. Insert KaiA and KaiBC into E. coli.
  2. Turn on KaiBC's promoter, thereby allowing for transcription of just KaiBC.
  3. Measure the amount of phosphorylated KaiC via Western blot.
  4. Turn on KaiA's promoter, thereby allowing for transcription of KaiA in addition to KaiBC.
  5. Measure the amount of phosphorylated KaiC via Western blot.

Ideally, we would see an increase in the amount of phosphorylated KaiC between steps 2 and 3. This would indicate that the Kai proteins are interacting in the same fashion as those within cyanobacteria. However, some care needs to be taken in deciding when exactly to measure the phosphorylated KaiC, in order to be able to discern the case where KaiC be actually be phosphorylated without the addition of KaiA. In that particular case, we may not see as much of a difference between steps 2 and 3.

Experiment 3

Goal
To measure the oscillation of the KaiC phosphorylation cycle, given spiked KaiA and KaiBC.
Procedure 
  1. Insert KaiA and KaiBC into E. coli.
  2. Turn on the promoters for KaiA and KaiBC at the same time, thereby allowing transcription of all three proteins.
  3. After a short time, turn off the promoters.
  4. Measure the amount of phosphorylated KaiC at certain intervals (i.e. every hour).

We hope to initially see strong oscillation, with the amplitude decreasing over time. Since we hope that no KaiA/B/C is being produced beyond the initial "spike", we would expect that the intracellular concentrations of the Kai proteins would decrease as the colony grows.

Experiment 4

Goal
To measure the oscillation of the KaiC phosphorylation cycle, given constant KaiA and KaiBC.
Procedure 
  1. Insert KaiA and KaiBC into E. coli.
  2. Turn on the promoters for KaiA and KaiBC at the same time, thereby allowing transcription of all three proteins.
  3. Measure the amount of phosphorylated KaiC at certain intervals (i.e. every hour).

We do not know how constant KaiBC transcription will interfere with the oscillation of KaiC (in cyanobacteria, KaiBC transcription oscillated on a circadian rhythm). This experiment should help us figure it out.

Experiment 5

Goal
To measure the oscillation of the KaiC phosphorylation cycle, given constant KaiA and spiked KaiBC.
Procedure 
  1. Insert KaiA and KaiBC into E. coli.
  2. Turn on the promoters for KaiA and KaiBC at the same time, thereby allowing transcription of all three proteins.
  3. After a short time, turn off the KaiBC promoter, leaving the KaiA promoter induced.
  4. Measure the amount of phosphorylated KaiC at certain intervals (i.e. every hour).

We would expect that, since KaiA is produced constantly in cyanobacteria, Experiment 5 yields slightly better oscillation than Experiment 3. However, we don't know for sure; like experiment 4, these predictions are highly speculative.


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