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

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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]

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
 * PCC 7942 + AM2195
 * PCC 7942 + modified AM2195
 * (2) with wild-type KaiABC knockout
 * Mutations
 * Mutate AM2195 for BB compatibility
 * Ligations:
 * 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
 * PCC 7942 + AM2195
 * (1) + modified AM2314
 * (2) + wild KaiABC knockout
 * Mutations
 * Mutate AM2314 for BB compatibility
 * Ligations
 * 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 = &alpha;(t) – &alpha;[P] [P](t) = [P](t – delta t) + &alpha;(t – delta t) – &alpha;[P](t – delta t) &alpha;(t – delta t) = sin(t) &alpha;(t) = k

A T: add &alpha; (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...



Incubator status

 * Added
 * PCC 7942 streak #1 plate
 * PCC 7942 streak #2 plate