AU: 7/27/2015

Here is the final kinetic parameter of WT P99 on Cephalothin which is very close to the previously published data. Now I will be doing kinetic analysis on Ampicillin and Ceftazidime.
Substrate: Cephalothin


Enzyme: WT P99 1 nM







Repeats
 Kcat (s-1)
 Vmax (µM/min)
 Km (µM)
 Kcat/Km (M-1s-1)
1
 137
 8.25
 9.95
 1.37 x 10^7
2
 141
 8.5
 10.15
 1.38 x 10^7
3
 104
 6.24
 7.52
 1.38 x 10^7
4
 128
 7.69
 10.22
 1.25 x 10^7
5
 130
 7.82
 10.32
 1.26 x 10^7

=


AU: 6/22/2015
I purified three batches of WT P99 enzyme and determined kinetic parameters using Cephalothin.
While purifying batch 1, there was a problem with cold room so I will ignore the values from this batch and will not use this enzyme further.
Both batch 2 and 3 looks good and Km values from batch 2 is slightly off from the batch 3. I plan to repeat the experiments in future but as of now, I will use another Amp and Ceftazidime so I have complete picture. My readings are close to published work (see attached doc file). Kinetic Data-Alok.docx. Once I finish this then I will start mutant purification and characterization.

AU: 6/22/2015- Updated MIC rank order- Rank order by MIC.doc
AU:6/10/2015:
I finished MIC determination, sequencing of all the clones I had in the library and got total 44 unique mutants of the library. Unique mutants as of 6-10-2015.doc. This include 8 single, 14 double, 16 triple, and 6 quadruple mutants.

I identified few higher MIC mutants but their sequencing data is not clear, possibly may be due to mixed colony (just speculation), so I will re-trasform these clones, A10, C1, C6, H7, and send them for re sequencing.

Meanwhile I am preparing for wild type purification and activity assay. In the literature, people have varied buffers in which they have purified protein so I am planing to have at-least two buffer for best activity.





AU: 6/4/2015:
Please data for Library2-Plate4: Library2-Plate4.pdf . The mutation information is not complete so I am not uploading at this time. I am waiting some repeat sequencing from Genewiz and upload accordingly.
Meanwhile, I need to repeat plasmid isolation from plate 3 which I will finish by Friday and send it to Genewiz for sequencing.


AU: 4/28/2015:
Total Mutants so far with MIC values, at the end of plate 3. I need to re-sequence some of the samples,but I would like to start plate 4. Once I finish plate 4 then I will repeat plate 3.

Rank order by MIC.doc (updated)

AU: 4/17/2015
I am attaching a really preliminary write up of Method section. I will keep updated once I go through again and again so it will be succinct and clear specially library construction section.
METHODS for Wiki 4-17-2015.doc



Q1: - for b-lactamase, why is IPTG concentration different in protein purification and in functional screening? Also, why different temperature, different timing of IPTG addition, etc
Answer:
There are three factor affecting protein expression (assuming clone is good).
1: The amount of IPTG
2: The temperature at which the protein is being expressed.
3: Timing of IPTG addition.

During functional screen, the idea is to provide saturating concentration of IPTG (1 mM or higher, and at 37 degree temperature) to see if the protein is being expressed or not. This concentration will ensure that protein will express from the clones as visualized on SDS-PAGE. But the quality of protein i.e. amount of soluble protein or in inclusion bodies (insoluble proteins present in the membrane) will be compromised. Since we are loading whole cell lysate, we are not concerned about soluble or inclusion bodies. Just need to see if protein is being expressed.

Growth Temperature before IPTG addition:
This will be 37 degree as it is optimum temp for E.Coli growth to get log phase growth. This temperature will be constant in all protocols.

IPTG addition time:
I have optimized the timing in such a way that after 2-2.5 hrs of inoculations, the bacterial cells will in in log phase (and have enough cells) to express protein. The log phase is defined as OD 600 = 0.4-0.5. This time doesn't vary much (may be +/- 15 min).

During purification:
We want to make sure that protein is in soluble for (or minimum insoluble protein/inclusion bodies), we use sub optimal IPTG concentration (in b-lactamase I chose 0.5 mM and it can be lowered, as some has reported). Same goes with the temperature, instead of 37 degree, I choose 25 degree to make sure that I produce soluble protein.

Q2: why do we use a different lysis reagent for purification.
Answer: Different proteins have different behavior in different lysis buffer. I chose the one from Cornish's paper, although I feel I may have to modify it depending upon my assay data. XG got lysis buffer from literature so her lysis buffer is different than mine. Lysis buffer is protein specific, not the method specific.

Q3: - For b-lactamase, His tag cleavage method not described?
Answer: For b-lactamase, I haven't seen any paper cleaving His-tag before assay/kinetics. Thus I assume that effect of his-tag on b-lactamase is nominal, if any. People have done studies with both His-tag and non-his tag protein.

Q4: - in sirtuin purification, I believe 2 rounds of His tag column purification are being planned; not for b-lactamase?
Answer: I assume that published Sirtuin published protocol has extra step to remove His-tag and that is why XG will cleave his-tag from final preparation. For b-lactamase, it may not be needed (please see answer of Q3).



AU: 4/1/2015:

Following clones in Lib2-plate 3 are +ve, clones are listed in 96 well format:
A1, A3, A5, A6, A7, A8, A9, A11
B5, B7, B8, B10, B11, B12
C2, C3, C4, C6, C7, C9, C10, C11
D1, D2, D4, D5, D6, D7, D8, D9, D11
E1, E3, E5, E7, E9, E10, E11
F1, F2, F4, F5, F10, F11
G3, G4, G6, G8, G9, G10, G11, G12
I will start MIC determination of these clones.



Data for Library 2 Plate 2: Library 2 Plate 2 data.pdf
Summary of mutants from Library 2 Plate 2: Lib2 plate 2 MIC-Mutant Info.docx
List of 37 Unique mutants so far: Unique mutants as of 4-1-2015.doc

AU: 3/24/2015:
Some papers related to this project in Dropbox:

C:\Users\aupadhyay\Dropbox\PMC-AT AU\Alok-Papers


AU 2/3/2015:
I am not uploading "protein gel" data. I get during IPTG screening. I have those pictures in digital format in computer attached to BioRad imaging system in the lab. If any one is interested, please ask, I will upload it here on wiki.

AU 1/29/2015:

1: Completed screening, sequencing, and MIC determination of two 96 well plates [See file named Library2-Plate1 page 1-7].
Key points:
a): 192 clones were screened for IPTG expression via SDS-PAGE analysis so far.
b): Out of 192, I got 28 unique mutants of the library.
c): In preliminary MIC determination, all 28 mutant showed less than WT MIC values.
d): Some of the sequencing were not perfect, that can be repeated once I finish prelim screening.

Library2-Plate1-Page1.PDFLibrary2-Plate1-Page2.PDFLibrary2-Plate1-Page3.PDFLibrary2-Plate1-Page4.PDFLibrary2-Plate1-Page5.PDFLibrary2-Plate1-Page6.PDFLibrary2-Plate1-Page7.PDF

2: Finished IPTG screening for Library2-Plate2 [See file Library2-Plate2-Page1].
Key points:
a): Total 52, out of 96, clones are IPTG induced protein expression positive.
b): I will start MIC determination and plasmid isolation from Monday.

Library2-Plate2-Page1.PDF
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

AU 1/12/2015
Library 2, plate 1 result for MIC, waiting for sequencing data and some MIC values on Amp.
MIC-Mutant-Lib2-Plate1.docx
AU 12/26/2014
Some of the new results, Library 2, plate 1. I will fill the table (total ~50 clones) as information comes in. MIC-Mutant-Lib2-Plate1.docx

AU 12/18/2014
Result:
MIC values for IPTG +ve clones (Library 2, Plate 1) on three different antibiotics: Ampicillin Cephalothin, and Ceftazidime. Result-Amp-Nov 26, 2014 (3).PDF MIC-Lib2-Plate1-Cephalo-Cefta.pdf
Since some of clones did not grow on Cephalothin and Ceftazidime, I will repeat this experiment just to make sure and confirm results.

AU 21/1/2014
Result: Combinatorial Library set 1 with approx MIC value with mutation information.

MIC-Mutant b-lactamase.doc

I am currently screening (IPTG expression and MIC values) with rest of clones. I have included another antibiotics, Ceftazidime, along with Amp and Cephalothin.


AU 10/9/2014:

I will update this tomorrow. I have to finalize data and transfer in power point. I got 7 more IPTG induced protein expression clones in the library.

AU 10/02/2014:
Beta-Lactamase library protein expression analysis.
B-lactamase library screen for protein expression.pptx

AU 9/19/2014:
Protein expression analysis result. Conclusions- Both WT P99 clones express well whereas mutants failed to express Beta-Lactamase on two IPTG concentrations (0.5 mM 2 mM)
Also, I did ligation transformation in BL21, got total 88 colonies, save the plate, need to do further optimization

Protein Expression WT and Mutants.ppt

AU 9/15/2014:
These files will be updated as I get more information/sequences.
Some of the plasmids need repeat sequencing and that can be decided after library is ready (this will avoid multiple primer design and synthesis. PLEASE feedback if you feel otherwise, Thanks)
I asked Genewiz for "free repeat": Reply "since reactions passed our QC, these reactions are not eligible for free repeats", Please feel free to request a "full price repeat" if you wish.

Raw Sequencing data.docAnnotated and aligned sequences.docLibrary Mutants.xls

RC (9-16):

1) Reviewed the docs above.
-Resequencing: will be done w/ same or different primers?
[AU: different primer]
-Regarding which mutant sequencing reactions are being repeated: AU2 reverse (are red highlighted amino acids incorrect?), AU3 forward, AU5 forward/reverseLibrary Mutants.xls
[AU: Updated library]

[AU: In both cases, AU2 and AU3, amino acid change is correct, if sequencing goes beyond 400-500 bases this problem comes where noise and signal is indistinguishable and its sequence curator's (Genewiz) call to assign a base. To make sure, in case of clone AU-2 the 5' sequences are good. This area was sequenced by R primer so in this case it would be reverse primer.
In clone AU-3, there is a stop codon just before authentic stop codon. Its same problem as in AU-2 clone. Since this area was sequenced by F primer so in this case it would be forward primer. Thus, for clone AU-2, Reverse Primer (New), for clone AU-3 it would be Forward primer (new).]

-Regarding which mutants are now being transformed in BL21: AU1-4 because AU5 appears to have extra mutations?
[AU: I feel that problem in sequence is due to sequencing error not the clone itself so I thought I will transform AU1-4, along with WT to evaluate expression. Since AU5 has extra mutation so no, I did not to transformation.]
-How many other sequences have you sent out?
[AU: so 36 clone but it will be 72 samples in total (since I am using 2 primers for one sample]

2) Discussion point: We may consider how to use sequencing data in experimental workflow, given that we are sequencing in advance of screening (unlike before):
[AU: I can try doing ligation transformation in BL21 and see if I get it, may require some optimization]

a) consider usefulness of having computational rank ordering of mutants for purpose of screening. or irrelevant given that we will be sequencing all 128 and that MIC screening is fast? depends on speed of screening based on MIC.
more useful in context of more time consuming protein purification and activity assays?

b) sequences in the current library have been generated based on mutations that appeared most frequently in the top ranked sequences from computations; all combinations of these mutations at the chosen positions were included in the library.
some of the combinations have already been computationally scored, others have not. if we want to use the scores during the experimental workflow, would need to pull up those scores that are available or score the sequences in combinatorial library that
were not yet explicitly scored. Any of these would require time from a computational staff member; would not be done before hiring of new programmer in ~ 1 mo.
note we only have scores for cephalothin at this time, but could later score other antibiotics
c) alternatively, look at correlations between MIC, activity/scores only after completion of experiments

3) We may consider outlining methods section of a manuscript on this project in the near future
a) Please post any papers CJ/AU have been referring to for library generation and screening methods for b-lactamase protein engineering (some may already be in CJ dropbox, but good to post on wiki for writing purposes)
b) if they used a similar protocol, was transformation efficiency of ligation product in BL21 reported to be low in this previous literature?

[AU: For number 2, we need discussion, I guess ( and I will give more thought to it once I have some free time.
I will update 3a. For screening, I think she planned using Cornish (2003).
For 3b, I doubt anyone report transformation efficiency of ligation in BL21 but in general, transformation efficiency of ligation product is low.]





AU 9/11/2014
AU 9/5/2014
Beta-Lactamase fragment information.
Beta-Lactamase fragments.doc

AU 8/26/2014
I have added "Task List from Alok" page. Thank you

CJ 3/14/2014
Gel analysis showed that the fusion PCR works well, generating products at ~1200bp, which is the desired length for P99 gene. After setting up the Tecan I will run the fusion PCR again with precisely measured substrate, so that the library can be constructed with minimal bias.
RC: From CJ's notebook, can you tell whether the latter fusion PCR with precisely measured substrate was done in order to prepare the library you are currently using?

CJ 3/10/2014
Sequencing results showed that the fragment 3 has also been correctly constructed. Now all three fragments are ready for the fusion-PCR assembly to construct the P99 library. I will do this after the Tecan arrives, so that DNA concentrations can be measured accurately.

CJ 2/21/2014
The fragment 1 and 2 have been successfully amplified. I also ran PCR to construct fragment 3. But the agarose in the lab was found to be expired and it forms insoluble clumps while heating in TBE buffer. Therefore I did not run gel to purify fragment 3. I have ordered new agarose and will gel purify f3 in the week of 3/3/14.

CJ 2/3/2014
The sequencing results for the second round of SDM have returned. Attached please see the alignment with wt P99 gene. We expect to see a TAT -> GCG mutation for Y241A; a TAT -> GGT mutation for Y241G; and a TAT -> TGG mutation for Y241W. All mutations were observed at the desired position.
IMAG0002.PDF


CJ 1/24/2014
The sequencing results for the first round of SDM have returned. Attached please see the alignment with wt P99 gene. We expect to see a TTA -> TAT mutation for L139Y; a TTA -> GAA mutation for L139E; and a TTA -> TGG mutation for L139W. All mutations were observed at the desired position.
Alignment for wiki.pdf


CJ 1/23/2014
Here I'm attaching a report explaining how I'm constructing the P99 library. It is in hand-written format because there are many drawings. As previously mentioned, I'm using a mixed SDM+fusion PCR strategy because it balances cost, work load, and quality. Currently I'm working on the first round of SDM on L139. I will post an update as soon as I receive the sequencing results.
P99 library design.pdf


CJ 12/17/2013
Attached below is my progress report for the past week on the beta-lactamase project. The bottom line is, I found the library was not correctly constructed. Therefore I don't see any point to proceed with it for growth assay or anything else. We must re-construct the library.
progress report 121713 CJ.doc

We can either have another company to do so, or I can try to do construct the library myself. There are two options: gene synthesis, or site-directed mutagenesis (SDM). I am familiar with SDM. I can also learn to do gene synthesis which is based on PCR and DNA ligation. It should not be very hard. I need to do more research on the cost of each method before deciding what to do.

RC: Thanks for the update. Good that you spotted this. Please proceed with looking into the construction of a new library.
RC (1-21): Are you preparing a library that is identical to the one prepared by BlueHeron, now using SDM, or are there differences in the library due to the change of method? As I recall the BlueHeron library used gene synthesis, making all possible combinations of selected mutations on DNA fragments that contained the residues that were subjected to mutation in the computational design.

CJ (1/21): I'm construction a library that is identical to the one from BlueHeron. However, I'm not using their gene synthesis methodology. Instead I'm going to do it via SDM in combination of fusion PCR. There are three reasons I'm not following BlueHeron's strategy: (1) their gene synthesis technology is based on solid-phase oligo synthesis and ligation, which is not available in our lab. (2) to do gene synthesis, we need to order >60 oligos, which would cost >$1000. (3) The error rate of gene synthesis is ~1 error per kb. The P99 gene is 1.2kb. This means if we use gene synthesis, every member in the library would probably bear one error. By contrast, SDM and fusion PCR are relatively cheaper and produce much less errors. BlueHeron employs gene synthesis because it is highly automated. But this is not a good option for home-made libraries.

RC (1-21): Ok, when you get a chance, please provide more details on your library construction protocol.