IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-6

From OpenWetWare

Jump to: navigation, search



Newer entries at the bottom.

Contents

Plasmid requests

We emailed Professor Golden and requested the following plasmids and their sequences:

  • pAM2195
    • Chloramphenicol resistance [NS 2.1]
    • PpsbAI::Lux AB [NS 2.1]
    • PpsbAI::Lux CDE [NS 2.1]
    • Ampicillin resistance [for selecting in E. coli, not on a neutral site]
  • pAM1579
    • Kanamycin resistance [NS 2]
  • pAM2314
    • Spectinomycin resistance [NS 1]
    • Streptinomycin resistance [for selecting in E. coli, not on a neutral site]

Game plans

Preface

Recall that we want to run two experiments in cyanobacteria:

  1. Control: A control experiment in which we insert the Lux AB/CDE genes under the psbaAI promoter (which is known to oscillate on a circadian rhythm), to callibrate our equipment and verify that the cyanobacteria's luminescence will oscillate. This will duplicate previous research.
  2. BB test: An experiment in which we knockout the wild-type KaiABC genes and insert our own BioBrick'd KaiABC genes, along with Lux AB/CDE from the control. This will verify that our KaiABC construct works.

Yesterday we came up with two plans for running these experiments.

Plan 1

Plan 1
                         PCC 7942 (unmodified)
                             /         \
                            /           \
       Control: + AM2195 [NS 2.1]      BB Test: + (AM2195 + BB'd KaiABC) [NS 2.1]
  • Plan 1 workload
    • Transformations
      1. PCC 7942 + AM2195
      2. PCC 7942 + modified AM2195
      3. (2) with wild-type KaiABC knockout
    • Mutations
      1. Mutate AM2195 for BB compatibility
    • Ligations:
      1. Ligate mutated AM2195 with BB'd KaiABC insert

Plan 2

Plan 2
                         PCC 7942 (unmodified)
                                 |
                                 |
                     Control: + AM2195 [NS 2.1]
                                 |
                                 |
                   BB Test: + (AM2314 + BB'd KaiABC) [NS 1]
  • Plan 2 workload
    • Transformations
      1. PCC 7942 + AM2195
      2. (1) + modified AM2314
      3. (2) + wild KaiABC knockout
    • Mutations
      1. Mutate AM2314 for BB compatibility
    • Ligations
      1. Ligate mutated AM2314 with BB'd KaiABC insert)

Comparison

Plan 1 has the advantage of running both experiments in parallel. However, we don't yet know whether neutral site 2.1 is large enough to accomodate the resistance cassete, LuxABC, and KaiABC (> 8kb size).

Plan 2 is conceptually cleaner, since we could reuse our control strain and keep separate functions on separate neutral sites. We also know that our inserts will fit. However, Plan 2 requires the control experiment to finish before starting the test (it's serial, not parallel).

Nick's suggestion

Nick suggested merging transformations 2 and 3 of each of the above plans by overwriting the wild-type KaiABC genes with our own BB'd KaiABC (plus resistance) via H.R., instead of knocking out the wild-type and inserting our genes at a neutral site in separate steps.

Nick's suggestion works better for plan 2 than for plan 1, since in plan 1 we would need to overwrite the wild-type KaiABC with our BB'd KaiABC plus Lux AB/CDE, which puts the Lux genes at a different position than the control, making the control less meaningful.

For plan 2, in contrast, we would be building on the control strain, so we wouldn't have to re-insert Lux at a different place.

Modeling

We want to make a model to analyze how the phosphorylation of KaiC changes as you add more KaiC.

The following are Nick’s ideas for the model:


D[p]/dt = α(t) – α[P]
[P](t) = [P](t – delta t) + α(t – delta t) – α[P](t – delta t)
α(t – delta t) = sin(t)
α(t) = k
A
T: add α (t – delta t) new objects to A
Remove (at random B length (A) objects from A
Update phosphorylated state of each member of A 
Calculate average phosphorylated states of A
[P] = [0,0,0]

Hydration of delivered primers

Peng's 10 primers for extracting and site mutagenesis of KaiABC came in; they were each rehydrated into 250uL of dH20 for a stock solution. Then, 20mM of each was made for use.

In the 250uL stock the concentration is:

 7942_KABC_extF: 94.08 nmol/mL
 7942_KABC_extR: 37.96 nmol/mL
 7942_KABC_crossF: 133.88 nmol/mL
 7942_KABC_crossR: 192.64 nmol/mL
 7942_KABC_pst1R: 154.16 nmol/mL
 7942_KABC_pst1F: 150.28 nmol/mL
 7942_KABC_pst2R: 153.64 nmol/mL
 7942_KABC_pst2F: 168.2 nmol/mL
 7942_KABC_eco1R: 145.84 nmol/mL
 7942_KABC_eco1F: 138.56 nmol/mL

To make a 20mM concentration with ~50 µL volume for each, add:

 7942_KABC_extF: 8uL stock, 37.63uL h20
 7942_KABC_extR: 16, 30.37
 7942_KABC_crossF: 6, 40.16
 7942_KABC_crossR: 4, 38.6
 7942_KABC_pst1R: 6, 46.28
 7942_KABC_pst1F: 6, 45.084
 7942_KABC_pst2R: 6, 46
 7942_KABC_pst2F: 6, 50.46
 7942_KABC_eco1R: 6, 43.75
 7942_KABC_eco1F: 6, 41.568

PCR from dead PCC7942

The goal here is to extract the kaiABC gene cluster from our dried out PCC7942 plates. Result: fail =(

Each person did 3 samples; 2 experimental and one control.

The experimental had:

 5uL template
 5uL 10x Buffer
 1uL dNTP (10mM ea) 
 1uL Primer (extF)
 1uL Primer (extR)
 0.5uL HotStarTaq
 36.5uL dH20

The run cycle was:

 #94C, 15'
 #94C, 30"
 #56C, 30"
 #72C, 1.5'
 #Cycle to step 2, 7x
 #94C, 30"
 #65C, 30"
 #72C, 1.5'
 #Cycle to step 6, 30x
 #72C, 5'
 #4C hold

Egel results show nothing good...

"didnt work so well...
"didnt work so well...

Incubator status

  • Added
    • PCC 7942 streak #1 plate
    • PCC 7942 streak #2 plate
Personal tools