Max -Rubisco

Question 1

what does rubisco stand for

Answer 1

ribulose-5-bisphospahte carboxylase oxygenase

Question 2

Rubisco is amde up of

Answer 2

8 large and 8 small subunits

Question 3

why are chaperones required

Answer 3

in vivo protein folding is very crowded requires assistance- molecular chaperones

Question 4

chaperones involved in protein folding

Answer 4

Bsd2
Cpn 60
Cpn10
Cpn20

Question 5

chaperones involved in protein assembly

Answer 5

RbcX
Raf1
Raf2
RbcL c terminus???????

Question 6

engineer pants rubisco in whar 3 ways

Answer 6

1. change aa (catalytic switches)
2. swap rubisco
3. swap rubisco + chaperones

Question 7

example of changes in aa in rubisco

Answer 7

2-5 aa replacement in rbcL gene by homologous recombination resulted in kinetic changes in tobacco plants

Question 8

example of swap rubisco

Answer 8

tobacco plant
substitute rbcL gene in chloroplasts +AB res gene via homologous recombination

• potato similar growth to WT, not better
• other rubiscos, cause plants not to grow - rubisco had not folded correctly - incorrect chaperones

greater the reduction in growth the greater the aa difference

Question 9

how do small su enter chloroplasts

Answer 9

cytosol nuclear genome rbcs gene . signal peptide directs proteni into chlorplast via tictoc complex
other chaperones also enter via tictoc complex

(large su + otehr chapeones encoded for in chlorplast)

Question 10

Cpn60 homolog?

isoforms?

Answer 10

GroEL

Cpn60 has two isoforms dont know which one is used

Question 11

Cpn10-20 homolog?

Answer 11

GroES

Question 12

Raf1 stands for? discovery?

Answer 12

Rubisco accumulation factor 1
discovered recently 2017 - by generation of random mutations by gamma rays
- rubisco was expressed but didnt function

Question 13

experiment by Whitney et al 2015 did what?

Answer 13

assembled foreign rubisco w/ native Raf1 gene

Tobacco +Arabid Rub +Arabid Raf1

co-expression with Raf1 increased foreign rubisco conten by >2 fold

BUT still lower than WT arabidopsis therefore ust be other chaperones

Question 14

experiment by Salesse-Smith 2018

Answer 14

overexpressed rubisco subunits and overexpressed Raf1 genes
-increase rubisco in C4 maize by agrobacterium mediated transfer

small su only = no increase in rubisco
small su + Raf1 genes = more rubisco –> have incrased growth compared to WT!!!

Question 15

increase in Rubisco in C4 does what

Answer 15

an increase from 12% to 14% soluble protein increased PS

oliver 2018

Question 16

increase in Rubisco in C3 does what

Answer 16

no inrease in PS
no physiologicl enhancement
metabolic load in C3 plant already met

is C:O2 limiting not amount of rubisco ????

Question 17

increase in Rubisco in algae does what

Answer 17

rubisco in algae is only 4% of soluble protein
increase PS rate

i dunno if it does or if this is twhat they want it to do

algae - biofuels, biomass, used for production of pharmaceuticals

Question 18

how to engineer Rubisco more easily and why?

Answer 18

plant transformation take ages

speed it up by assmeblying in e. coli

Question 19

study by Aigner did what?

Answer 19

engineer in e. coli
3 x plasmid 
1 - RbcL    (araB)
2- Cpn60/Cpn20    (T7)
3- Raf1/ RbcX, Bsd2    (T7)

diff promoter, T7 induced by sugars, incrased levels of chapeones before expressing rbcL gene
araB induced by arabinose in media

Results:

• PAGE-GEL results; confirmed requirements of each chapeone
• all 3 expression gave the highest rubisco expression in e.coli
• some worked even without Cpn20 - this enzyme is homologous to GroES, must be similar enough to substitute one another

Question 20

what chapeones are general

Answer 20

Cpn60/20/10

Question 21

what chapenones are rubisco specific

Answer 21

RbcX, Raf1, Raf2, raf3, Bsd2 etc

Question 22

Study by Lin et al 2019 did what

Answer 22

modified study by Aigner 2017, to express tobacco genes and motiored chapeone + rubisco kinetics

altered small subunits used

the native to tobacco trichome small subunit has a higher catalytic rate but lower CO2 affinity compared to the other small subunits tested

tobacco rubisco successfully expressed and assembled in e.coli using PET and pCDF expression systems