shanghai dogs are very special!

Shanghai dogs are very special!

There are many dogs in ShanghaiXuhui JTU campus

Hypothesis ：Shanghai dogs can read 汉字 (and obey orders)

Result ：The clever dog 狗 recognized the label 肉 and ignored the box labeled 菜Conclusion: At JTU even the dogs are clever

What was good?

Any problems?

What should we learn?

What was good?

Good observation

Great idea!

Any problems?

鬣狗 hyenas are not dogs

How can you find out whether the bacterium that is growing in your flask is the correct one?Discuss

老托的故事 I have seen many scientists wasting time because they failed to recognize contaminants.

How to detect contaminants?• Streak out for single colonies

• (Phase contrast) microscopy

• Test for known markers (e.g.

antibiotic resistance)

• Use specific antibodies etc.

• PCR (only on pure cultures)

• Check early and keep checking!

PNAS 2001 vol. 98/14

The extent of cell line contamination is enormous.

45 out of 252 (1/5) new cellcultures deposited at a German cell line repository were contaminated by another cell line.

Hundreds of scientific reports basedon fraudulent [wrong] cell lines were published.

Any problems?

Living things have several

senses to ‘read’ the

environment

List all the senses you know, and the organisms where they are most important. Start with bacteria, give examples.Try to explain without using the dictionary

12

3

4

5

1 12

2 2 2

If you repeat an experiment, make sure that it is a true, independent repeat

Does a specific mutation increase antibiotic production?Wild-type strain 0

Mutant strain 1 Mutant strain 2Clonal copy of 1

Gene replacements to generate identical mutations

mg ml-1A B C D E F G H I K L M

Measuring antibiotic production (bioassay, LC)

Growing the bacteria (culture broth/medium, flask/fermenter)

1. Which values will be most similar (small SD, standard deviation)?2. Which measurements represent independent repeats?3. The 3 mutant strains should be the same; why could we get very

different measurements?

Group 1 Group 2

• The values from Group 1 and Group 2 could be very different if one of them had suffered an additional spontaneous mutation.

• Additional spontaneous mutations are common if a gene replacement causes the cells to grow slowly because Compensatory mutations may make the strain fitter.

• All the values in Group 1 are dependent on each other because they all go back to mutant 1

• Only measurements from Group 1 measurements from Group 2 qualify as independent repeats

Streptomyces coelicolorMutants

How to prove that the mutation you created causes the observed phenotype?

Genome

Wild-type gene

Introducing a specific mutation into the genome by gene replacement

Genome

Planned, site-directed

mutation

The wild-type gene was replaced by a mutant

gene or deleted completely by replacement

e.g. with a selectable antibiotic resistance gene

How to confirm that the observed phenotype was caused the mutation you created,

and not by a surprise spontaneous mutation somewhere else in the genome ?

Genome

Planned, site-directed

mutation

I there another spontaneous

mutation?

What experiment(s) could you do to find out?

• explain on paper using a diagram with minimal text.

How to confirm that the observed phenotype was caused the mutation you created, and

not by a surprise spontaneous mutation somewhere else in the genome ?

Complement the mutation in-trans with a copy of the wild-type gene

Genome

Planned, site-directed

mutation

I there another spontaneous

mutation?

Wild-type gene

Plasmid

in-trans means ‘somewhere else’

cis complementation means repairing the mutated gene itself

1. Interpretation of experiments

2. Contamination of cell cultures

3. Independent repeats

4. Spontaneous mutations

5. trans complementation