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T-Wisse / MEP_Thomas

This repository serves as the documentation platform for my MEP in TU Delft.
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SATAY on transformed yTW001 and yLIC137 #19

Closed T-Wisse closed 3 years ago

T-Wisse commented 3 years ago

Preparation: To do SATAY we require several different liquid media as well agar plates. These will have to be prepared beforehand to save time. I will look up what we need and note it down below.

Hypothesis (We believe that...) We believe that we can induce the transposon in the plasmid for many cells of our strains yTW001 and yLIC137 and allow them to grow for a number of generations (=perform SATAY) as detailed by benoit.

Test (To verify that we will...) Perform the protocol using our clones observed on the -Ura plates from the sanity check.

False Test (To falsify that we should test...) Metric (And measure...) We measure OD and measure the cells/ml as well as ADE+ cells/ml and URA+ cells/ml by plating throughout the preculture, induction and reseed steps.

Criteria (We are right if...) We are right if we observe a increase in ADE+ cells during induction and reseed similar to described by benoit. (% end of induction?). At the end of the reseed ~70% of cells should be ADE+ and expect a pellet of about 15ml.

Observation (We observed...) We observed an increase of ADE+ cells during induction, with 0.075 to 0.27% of ADE+ cells at the end of induction. At the end of the reseed the % of ADE+ cells ranged from 25% to 100% or 35% to 75%, depending on which plates are used to determine cells/ml. The pellet obtained was slightly lower than 15 ml.

Learnings and insights (From that we learned that...) We have performed SATAY with some succes

Decisions and actions (Therefore we will...) Continue by extracting the DNA of the pellet and preparing it for sequencing.

T-Wisse commented 3 years ago

[In progress]

Based on SATAY paper Michel et al. 2017 and previous SATAY experiments of Leila

leilaicruz commented 3 years ago

Induction: SD-URA+2% gal (+ade?)

Yes with adenine

Measure background induction (essentially sanity check but more numeric)

it is not the same since you wont analyze colonies but the whole culture background before induction

Plating in SD-ADE and SD-URA to check induction

and also in SC complete media