Biomod/2013/Harvard/lab: Difference between revisions

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The image below shows all of the GLucCaM and GLuc constructs that we have created by the end of Week 2. They were all inserted into the pDIMC8 backbone, which contains a chloramphenicol resistance gene, a multiple cloning site, and origin of replication, to create a plasmid that can be expressed by ''E. coli''.  
The image below shows all of the GLucCaM and GLuc constructs that we have created by the end of Week 2. They were all inserted into the pDIMC8 backbone, which contains a chloramphenicol resistance gene, a multiple cloning site, and origin of replication, to create a plasmid that can be expressed by ''E. coli''.  


[[Image: GLucCam_Plasmids_Week2.png|thumb|left|300px|GLucCaM Plasmids]]
[[Image: GLucCam_Plasmids_Week2.png|thumb|left|275px|GLucCaM Plasmids]]
[[Image: GLuc_Plasmids_Week2.png|thumb|right|300px|GLuc Plasmids]]
[[Image: GLuc_Plasmids_Week2.png|thumb|right|275px|GLuc Plasmids]]


Sequencing results of the GLuc plasmids were good
Sequencing results of the GLuc plasmids were good
Line 153: Line 153:
*We are doing the assembly in two steps because Gibson assembling them all at once did not seem to work
*We are doing the assembly in two steps because Gibson assembling them all at once did not seem to work
**This should be a more time-consuming, but more effective process (and hopefully successful)
**This should be a more time-consuming, but more effective process (and hopefully successful)
*Sequencing confirmed successful assembly of BlaCaM-AIDA and CaM-AIDA
**Bla-AIDA is being resequenced over the weekend.
*We seem to be good to start attempting to display our proteins on the cell surface.
We have a few methods to test the effectiveness of our display
*We will add trypsin to the cell supernatant, digesting any displayed proteins
**Before and after gels should show the disappearance of our displayed protein
*We will use either benzyl-penicillin or CENTA substrate to test beta-lactamase activity in the cell supernatant
**Cells with displayed Bla should show reactivity with benzyl-penicillin or CENTA
*We are performing a series of assays to determine how membrane-permeable CENTA is
**Hopefully it does not travel freely into cells, and is therefore suitable to test with our displayed proteins
***Because of its easily measurable color changes upon reaction with beta lactamase, CENTA is a very good substrate to test with
**Benzyl-penicillin has been shown to no permeate the cell membrane well, so that is a fallback option
***It does undergo such measurable changes when it is hydrolyzed, but there are reports of being able to measure the concentration via UV-vis, provided all cell matter is spun down first.
*Initial assay showed less CENTA hydrolysis in the whole cells, but there was still a significant color change.
**This could be a result of membrane permeability to CENTA, or the presence of lysed cells in our whole cell solution, or some yet undiscovered reason.
**More tests will have to be done.
==Week 5==
Resequencing of Bla-AIDA constructs confirmed a successful assembly, so we are ready to move ahead with expression.
Expressed at 37°C with .5, 1, 2 mM IPTG as per [[Biomod/2013/Harvard/references#Input_Domain | Latteman, et al.]]
Also expressed at 25°C overnight to have fresh cells for assays.
None of these conditions produces positive results.
*No visible bands on gels
**Compared various expression conditions and looked for evidence of trypsin digestion of displayed proteins
**None confirmed expression
*No activity on multiple CENTA assays
**Whole-cell β-lactamase tests were not above baseline.
**Some problems with the plate reader and the high-density of our cells, but there is still likely zero activity here.
Need to find a way to get these genes to express soon!
==Week 6==
Began mutagenesis on BlaCaM
*Error-prone PCR on just the CaM part of the fusion
**Then we will reinsert into a vector containing the two halves of Bla
*Even if we cannot get display working, we can still do screening on lysates of BlaCaM with CENTA.
**Basically just run many, many assays on BlaCaM mutants looking for increased affinity.
**Since we cannot use selection, this is not nearly as high throughput, but we should still be able to adapt BlaCaM to a new substrate.
*We will continue to work on the display for the time being, but it's good to have a fall back for now.
*We are now going to try expressing our constructs in a DH5α-derived strain, as that the strain that [[Biomod/2013/Harvard/references#Input_Domain | Latteman, et al.]] has had success with.
Screening of library was inconclusive at first.
*Not familiar with protocol for lysate screening, so I ended up with some unreadable plates
**Maybe also expressed the proteins for too long
*Will try this all again next week now that I have a handle on what to do...
==Week 7==
Final attempts at getting the AIDA display working
*Expression in DH5α
**Trypsin digest assay was inconclusive
*No hard evidence that the display is working
*No conclusive bands on gels
*Probably time to give up working with bacterial display..
Screening of lysates is also not going great
*Looks like a lot of non-specific binding going on in the lysates
**Signal is getting drowned out
**Following the lysate screening assay from Glenna's paper exactly yields no results
*We are going to move to screening 6xHis purified libraries
==Week 8==
We got purification columns and plates
Picked from our BlaCaM library plates in 96-well plates
*With our purification plates we should be able to screen 96 mutants at once
This worked well, we saw good M13 activity and lower d-toxin
*This is like what we saw with the purified WT BlaCaM at the start of the summer
Sequencing shows a low error rate (1-2/gene)
*We wanted a higher error rate so we will make some new libraries with more errors
==Week 9==
PCR'd some more BlaCaM libraries with error-prone PCR
Also ironed out some problems with our 96-well screening protocol
Now use lysozyme and nuclease during lysis to help keep viscosity low
*This really helps our lysates go through the spin columns better
Our assay on our first 96-well purification showed no exciting hits
*Need to see what the higher error rate produces
This week was slowed some because we accidently were working with a construct that didn't have a His tag
*Oops
==Week 10==
Lots of picking of libraries, expression, purification, and assays this week
New libraries have 3-4 errors/gene (~6-8/kb)
Assay on 3rd library (first with high mutation rate) was good!
*Definitely some higher d-toxin activity in some mutants
We use the metric: d-toxin/M13 -  negative control/M13 to measure how much our mutant has improved it's d-toxin binding
*Basically measuring what fraction of the d-toxin signal is attributable to new d-toxin activity, versus just background activity
*How close does the d-toxin signal get to the positive control?
*Because we are not controlling directly for protein concentration, this metric does some correction for expression level and protein concentration by computing signal relative to the M13 positive control
Lastly, we took some hits from earlier libraries and expressed them in higher volume
*We will assay these in triplicate to confirm them
*Also created a few more libraries that will be frozen for use during the fall
**But probably more useful to take the hits from our first 4 libraries and do a 2nd generation on them
==Sept-October work==
Most of September spent confirming hits from previous libraries
*using our d-toxin/M13 -  negative control/M13 metric on hits repeated in triplicate removed a lot of them
*Most hits were not repeatable - sort of expected
*We did identify 2 possible improvements though: C12 from library 3-1 and H3 from library 3-2
**They display about 60% and 300% improvement in d-toxin activity over the wild-type.
**We will do a 2nd generation on these hits and hopefully get some more d-toxin activity!

Latest revision as of 08:41, 30 September 2013

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Laboratory Notebook

Week 1

6/10/13

General BioMOD Cloning

purpose: clone for DH5α pDIMC8 BlaCaM His and DH5α pDIMC8 DsbA BlaCaM
  1. 5 mL LB
  2. 100µL 20% glucose
  3. Cm (34 mg/mL working stock), 7.35 µL of this, final concentration 50 µg/mL
  4. Add cells
  5. Inoculate overnight

6/11/13

I. Bacterial Solution: LB Agar Plates

purpose: to make agar plates for 1L LB-agar
  1. Mix stock
    1. 960mL DI water (860 mL if using glucose optional)
    2. 10g of Tryptone
    3. 10g of NaCl
    4. 5g yeast extract
    5. 15g agar
  2. Autoclave
    1. liquid (not dry) 30 min. sterilization/20 min drying
    2. cool to ~50°C in water bath
  3. Done under flame
    1. Add glucose - final concentration 20% glucose, if using 20% glucose then 100 mL, optional
    2. Add antibiotics to the specified final concentration. e.g. Cm: 50 μg/mL. Working stock: 34 mg/mL ⇒ 735 μL per 500 mL
    3. Pour onto the petri dishes, pre-mark them by type, e.g. : two black lines: Cm, red line: glucose
    4. Store

II. Experiment II from 6/10/13 continued: Miniprep (QIAGen)

purpose: to purify plasmids from our clone colonies
We followed MiniPrep
  1. transfer cell cultures into centrifuge tubes. Be careful not to drop the tip in.
  2. Centrifuge for 5 min. at 4000 rpm. N.B. make sure things are balanced.
  3. Dump the supernatant into old tube. Later add bleach before pouring down sink.
  1. Resuspend pellets and tansfer to 1.5mL labeled tube with 250μL P1 Buffer.
  2. 250 μL P2 Buffer (blue color): mix thoroughly.
  3. Time since step 2 must be less than 4 minutes: add 350μL of N3 Buffer. Mix thoroughly.
  4. Centrifuge at 13,000 rpm for 10 minutes.
  5. Apply the supernatant to QIAprep spin column.
  6. Centrifuge 1 minute. Discard flow-through.
  7. Wash w/ 750 μL Buffer PE.
  8. Centrifuge 1 minute. Discard flow-through.
  9. 1 min additional centrifuge.
  10. Column into 1.5 microcentrifuge tube.
  11. Elute with 50 μL water. 5 minute rest.
  12. Centrifuge 1 minute.

III. NanoDrop

We followed NanoDrop

IV. PCR Amplification: HercII PCR

Purpose: make backbone vectors for our Gibson reaction from pDIMC8 DsbA BlaCam. Will make:
  • pDIMC8 AIDA
  • pDIMC8 DsbA GLucCaM
  • pDIMC8 GLucCaM His
  • pDIMC8 DsbA GLucCaM His

Forward/Reverse Primer Chart:

Forward Reverse Key
AIDA pDIMC8 gib - for pDIMC8 gib - rev FW
DsbA GLucCam pDIMC8 gib - for pDIMC8 DsbA gib - rev CE
GLucCaM His pDIMC8 addHis - for pDIMC8 gib - rev TRN
DsbA GLucCaM His pDIMC8 addHis - for pDIMC8 DsbA gib - rev DZ

We followed Herc II PCR protocol.

Week 2

Transformed and grew up colonies of cells with CE, TN, and DZ plasmids.

  • Realized that our TN plasmids were assembled with incorrect primers.
    • The resulting plasmids almost certainly do not have the genes we want inserted.
  • Used Qiagen MiniPrep kit to extract DNA from cells
    • Sent DNA off to have our plasmids sequenced to check for correctness.
    • Only send a few colonies of DZ and CE cells -- TN was not worth checking

Results:

  • The DZ plasmid was correctly assembled in one of our sequences
    • The other had a single mutation at residue 31 of GLucCaM gene
  • One of the CE sequences was confirmed as well
    • The other produced a sequence of too low quality -- likely not a correct plasmid

We started work on GLuc plasmids that contain only the G-Luciferase gene without the Calmodulin insertion. These will be used as controls during our future experiments.

  • Assembled plasmids with the same combination of His and DsbA tags as our GLucCaM plasmids.

The image below shows all of the GLucCaM and GLuc constructs that we have created by the end of Week 2. They were all inserted into the pDIMC8 backbone, which contains a chloramphenicol resistance gene, a multiple cloning site, and origin of replication, to create a plasmid that can be expressed by E. coli.

GLucCaM Plasmids
GLuc Plasmids

Sequencing results of the GLuc plasmids were good

  • Only the EF construction failed to return a correct sequence
    • Sent off 8 more colonies of EF for sequencing in the hope of finding a correct plasmid

The following table summarizes the progress we have made thus far with the GLucCam and GLuc plasmids.

Received AIDA autotransporter gene and began construction of plasmids containing AIDA and AIDA+insert (Bla, CaM, BlaCaM) fusions for bacterial display.

Week 3

To start off the week, we received our sequencing results for EF from Friday and found that the EF2-2, EF2-4, EF2-8, and EF2-6 samples had been successfully transformed with only noncoding mutations ( @902 A insert; @1017 G -> A mutation)

Continued construction of bacterial display plasmids.

  • Lots of assembly to be done here.
  • The gene that we want displayed must be inserted after the signal sequence and before linker region of the AIDA construct.
    • All of that must be put within our pDIMC8 vector.
  • This week was spent mainly trying to resolve cloning difficulties while creating our display plasmids.
    • Attempts at a 4 part assembly failed, and we also had to reconstruct a vector that refused to be modified.
  • We hope to have AIDA-Bla/CaM/BlaCaM fusions completed by the end of the short week next week.

We also began discussing how we will perform directed evolution once we have our display functioning.

  • Consensus seems to be on ordering an error-prone PCR kit because of its speed and ease of use

Week 4

Input: Our current focus is inserting Bla/CaM/BlaCaM into our backbone, which is the AIDA gene split and fused into the pDIMC8 vector

  • We are doing the assembly in two steps because Gibson assembling them all at once did not seem to work
    • This should be a more time-consuming, but more effective process (and hopefully successful)
  • Sequencing confirmed successful assembly of BlaCaM-AIDA and CaM-AIDA
    • Bla-AIDA is being resequenced over the weekend.
  • We seem to be good to start attempting to display our proteins on the cell surface.

We have a few methods to test the effectiveness of our display

  • We will add trypsin to the cell supernatant, digesting any displayed proteins
    • Before and after gels should show the disappearance of our displayed protein
  • We will use either benzyl-penicillin or CENTA substrate to test beta-lactamase activity in the cell supernatant
    • Cells with displayed Bla should show reactivity with benzyl-penicillin or CENTA
  • We are performing a series of assays to determine how membrane-permeable CENTA is
    • Hopefully it does not travel freely into cells, and is therefore suitable to test with our displayed proteins
      • Because of its easily measurable color changes upon reaction with beta lactamase, CENTA is a very good substrate to test with
    • Benzyl-penicillin has been shown to no permeate the cell membrane well, so that is a fallback option
      • It does undergo such measurable changes when it is hydrolyzed, but there are reports of being able to measure the concentration via UV-vis, provided all cell matter is spun down first.
  • Initial assay showed less CENTA hydrolysis in the whole cells, but there was still a significant color change.
    • This could be a result of membrane permeability to CENTA, or the presence of lysed cells in our whole cell solution, or some yet undiscovered reason.
    • More tests will have to be done.

Week 5

Resequencing of Bla-AIDA constructs confirmed a successful assembly, so we are ready to move ahead with expression. Expressed at 37°C with .5, 1, 2 mM IPTG as per Latteman, et al. Also expressed at 25°C overnight to have fresh cells for assays. None of these conditions produces positive results.

  • No visible bands on gels
    • Compared various expression conditions and looked for evidence of trypsin digestion of displayed proteins
    • None confirmed expression
  • No activity on multiple CENTA assays
    • Whole-cell β-lactamase tests were not above baseline.
    • Some problems with the plate reader and the high-density of our cells, but there is still likely zero activity here.

Need to find a way to get these genes to express soon!

Week 6

Began mutagenesis on BlaCaM

  • Error-prone PCR on just the CaM part of the fusion
    • Then we will reinsert into a vector containing the two halves of Bla
  • Even if we cannot get display working, we can still do screening on lysates of BlaCaM with CENTA.
    • Basically just run many, many assays on BlaCaM mutants looking for increased affinity.
    • Since we cannot use selection, this is not nearly as high throughput, but we should still be able to adapt BlaCaM to a new substrate.
  • We will continue to work on the display for the time being, but it's good to have a fall back for now.
  • We are now going to try expressing our constructs in a DH5α-derived strain, as that the strain that Latteman, et al. has had success with.

Screening of library was inconclusive at first.

  • Not familiar with protocol for lysate screening, so I ended up with some unreadable plates
    • Maybe also expressed the proteins for too long
  • Will try this all again next week now that I have a handle on what to do...

Week 7

Final attempts at getting the AIDA display working

  • Expression in DH5α
    • Trypsin digest assay was inconclusive
  • No hard evidence that the display is working
  • No conclusive bands on gels
  • Probably time to give up working with bacterial display..

Screening of lysates is also not going great

  • Looks like a lot of non-specific binding going on in the lysates
    • Signal is getting drowned out
    • Following the lysate screening assay from Glenna's paper exactly yields no results
  • We are going to move to screening 6xHis purified libraries

Week 8

We got purification columns and plates Picked from our BlaCaM library plates in 96-well plates

  • With our purification plates we should be able to screen 96 mutants at once

This worked well, we saw good M13 activity and lower d-toxin

  • This is like what we saw with the purified WT BlaCaM at the start of the summer

Sequencing shows a low error rate (1-2/gene)

  • We wanted a higher error rate so we will make some new libraries with more errors

Week 9

PCR'd some more BlaCaM libraries with error-prone PCR Also ironed out some problems with our 96-well screening protocol Now use lysozyme and nuclease during lysis to help keep viscosity low

  • This really helps our lysates go through the spin columns better

Our assay on our first 96-well purification showed no exciting hits

  • Need to see what the higher error rate produces

This week was slowed some because we accidently were working with a construct that didn't have a His tag

  • Oops

Week 10

Lots of picking of libraries, expression, purification, and assays this week New libraries have 3-4 errors/gene (~6-8/kb) Assay on 3rd library (first with high mutation rate) was good!

  • Definitely some higher d-toxin activity in some mutants

We use the metric: d-toxin/M13 - negative control/M13 to measure how much our mutant has improved it's d-toxin binding

  • Basically measuring what fraction of the d-toxin signal is attributable to new d-toxin activity, versus just background activity
  • How close does the d-toxin signal get to the positive control?
  • Because we are not controlling directly for protein concentration, this metric does some correction for expression level and protein concentration by computing signal relative to the M13 positive control

Lastly, we took some hits from earlier libraries and expressed them in higher volume

  • We will assay these in triplicate to confirm them
  • Also created a few more libraries that will be frozen for use during the fall
    • But probably more useful to take the hits from our first 4 libraries and do a 2nd generation on them

Sept-October work

Most of September spent confirming hits from previous libraries

  • using our d-toxin/M13 - negative control/M13 metric on hits repeated in triplicate removed a lot of them
  • Most hits were not repeatable - sort of expected
  • We did identify 2 possible improvements though: C12 from library 3-1 and H3 from library 3-2
    • They display about 60% and 300% improvement in d-toxin activity over the wild-type.
    • We will do a 2nd generation on these hits and hopefully get some more d-toxin activity!
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