Revision as of 12:01, 1 November 2017

Sponsors

Contact

Preparation

Preparation

16/3/17

Autoclaved items and prepared media

17/3/17

Obtained S. cerevisiae BY4741 from Dr. Rajyaguru’s lab and incubated it at 30 degree celcius

Inoculated the above strain in 2 mL YPD [4.5 mL YPD media + 0.5 mL 20% glucose] using a sterile toothpick

18/3/17

Went to Prof. Dighe’s lab to get some chemicals, sans people

Yeast in the inoculated tube are growing fine but seem to have aggregated at the bottom.

19/3/17

Started 8 growth curves

Cuvette placement (orientation) was not right for the first few readings; path length was not 1 cm.

21/3/17

Autoclaved plates, falcons, media (YPDA and YPGA)

Got the plasmid pRS316 and HeLa cells from a lab

Decided to try shockwave transformation of yeast. We will be amplifying the plasmid pRS316 in E. coli, mini prepping it and taking it to Aerospace department.

22/3/17

Got DH5a competent cells from Prof. Chakravortty’s Lab. We have transformed the pRS316 plasmid in E. coli. We also made SOB media. Our selection marker for now is Ampicillin.

23/3/17

Plates (one streak and one spread) yeast culture and kept the remaining in cold room. We also observed plates of E.coli DH5a today

24/3/17

Transformants from the Amp+ plate were inoculated in SOB (3 test tubes)

WT from the amp- plate was inoculated in SOB (2 test tubes)

Observations: No GFP fluorescence was observed, possibly because plasmid is low copy (?). Note from Sai: In hindsight, we should have realized that the GFP was under a yeast promoter (hehe)

27/3/17

Step 8 of protocol failed: No pellet was obtained. Need to redo entire set of experiments :(

Miniprep was then carried out (protocol follo9wed from Sambrook

29/3/17

Plasmid (Yeast GFP) brought from Prof. Rajyaguru’s lab

pRS316 Amp/Ura. High conc, use 1 μL from Nupur’s protocol

Will show GFP expression in yeast only, not E.coli

31/3/17

Buffers for electrophoresis were made and autoclaved

Electrophoresis showed presence of plasmid! :D

02/4/17

YPD media prepared and inoculation done.

03/4/17

Showckwave transformation canceled by Akshay. Images of yeast cells were taekn under the microscope for heamocytometry

At night comp cells prep was done. Gel was run for the transformants (terrible results, needs redoing)

30/4/17

Lab meeting done at 20:00. Plans were laid down for the whole project. As a whole, need to

Overexpress hexose transporters
Overexpress invertase
ACE2 gene knockout to induce clumping
Create GFP-GVNP and GVNP-TEV biobricks
Hardware part
Fiji plugin for haemocytometer yeast count

Fresh LB, YPAD, YPAD (2x) was prepared

01/5/17

Heat shock transformation carried out in Dipa lab.

02/5/17

(DC lab) Observation:- The colonies look good. Thus procedure shall be taken forward. Fresh media prepared, plates autoclaved.

04/5/17

Raj and Sharath arried out miniprep in DC lab.

Nanodrop readings too high. Ran, gel; too thick, not too clear. Repeated gel with lower concentration of DNA, presence of genomic DNA contamination and RNA contamination (probably non functional RNAse), need to repeat experiment

05/5/17

Raj and Sharath repeat miniprep, ran gel. Do get plasmid bands but with heavy DNA adn RNA contamination (except for the third lane, for reasons unknown)

SOB plates were prepared, YPAD plates were prepared, and some required reagent solutions were prepared

07/5/17

YEast cultures discarded, cant store them properly without drastically affecting the effciency. Fresh incoculum prepared

08/5/17

Bulk extraction commences from a 50 ml culture. Ran the gel and SUCCESS! :D . Realized some errors in our protocol which helped us prevent genomic DNA contamination. Nanodrop readings were positive and resuspension was done in TE and not MilliQ.

09/5/17

Met Nupur and asked for selection media, and the compelte plasmid map we were given. Alos shockwave transformation is a go, the first trial shoould be over tomorrow, 10th

10/5/17

Cleaned up the lab: hoods, drawers, trash. Rearranged equipment more sanely. Checked stocks of Eppendorfs, tips, etc.

11/5/17

LiAc transformation and shockwave transformation were carried out. Technical difficulties lead to LiAc transformation taking a LOT of time to carry out.

Media was prepared

After meeting with Dr Srinath, realized "Why work with yeast? E.Coli is easier to work with, protocols are easier and less time consuming"

12/5/17

Made reagent stocks.

15/5/17

Media preparation. Different percentages of agarose gel prepared to check for fragility and suitability for low melting agar for cell density measurements. The burette idea was discarded, because vortexes for amd mixing occurs.

16/5/17

Inoculated DH5A (no plasmid) in LB media. Realized there are some contamination problems. Glycerol stocks were prepared and some transformants were plated

18/5/17

More inoculations done, growth curve of E.coli and yeast was carried out.

At 4:35 PM Srinath sir entered the lab and questioned everyone about the project. He pointed out various errors people were committing and asked for a lab meeting scheduled on 19th.

30/5/17

The lab notebook had been neglected for the past few days, sadly

Yesterday, five different transformants of pSB1A3 (ampicillin resistant) were plated. Today, we will miniprep the plasmid and ideally run a gel to confirm. To do this, we have prepared alkaline lysis solutions I and III beforehand.

Made workling stocks of the required reagents

Ran a miniprep. Ran a gel, absolutely no bands were present. There was a minor screwup while loading. Will run gel again tommorrow.

05/6/17

Once again, a major ga in the lab notebook.

Possible reason for failure in gel- maybe used 50x TAE rather than 1x. Maybe EtBr diffused too fast. The second gel ran on 31st failed because probably 20x SB was used. . Even the ladder was not seen so the problem lied with the gel. The third gel maybe ran for too long and thus no bands were seen.

On the plus side, the assembly plan is going well. Primers are being designed, to be finalized with Arunavo tomorrow and then with Srinath sir afterwards

Four primary inoculums made today. If they are dense enough then we'll carry out miniprep tomorrow, then run a gel and discuss primers.

08/6/17

Haemocytometry imaging was carried out. We tried out Nigrosin staining tog ive contrast between cells. There was non uniform cell density due to smearing and dead cells take up the die,thus we need to counterstain with something else. We need to come up with aa reproducible protocol for collab with other teams

LB kits and SOB kits were made. Comp cells were tranformed using pSB1C3 and pSB1A3.

09/6/17

Imaging was tried using the saturated e.coli and yeast primary inoculum (set up on 8.6.17 night). Nothing significant came out from the 1st sem lab compound microscope for safranin staining.

Phase contrast inverted microscope was used to take images. The cells and the grid of the haemocytometer do not come on the same focal plane. So superimposition of the cell and the grid image was suggested. Also E.coli cells doesn't come in 10X zoom and in 40X zoom the grids of the haemocytometer go out of the frame. ~Will try to take image from the inverted microscope on Monday (12.6) in 1st sem lab.

Plates containing transformed cells didnt show any colonies till night

10/9/17

SOB+chloramphenicol plates are made for transformation. Cells were transformed with 10, 50, 100 pg/microL concentrations of the plasmid and plated on SOB+ chloramphenicol plates in duplicates .Two controls were plates on -ve chloramphenicol and +ve chloramphenicol plates.

11/6/17

In the morning, imaging was done with safranin. Not going to use nigrosin anymore. Tried E. coli and yeast. Yeast looks good without dye, but planning to use it anyway to improve contrast for the software. Camera takes worse images compared to direct view through eyepiece. :(

Prepared more SOB plates with and without chloramphenicol. Went to Dighe’s lab, collected 3 aliquots of competent DH5alpha and two plasmids (NRR3GST and N3NRRCisT). Will collect Pichia culture/plate from Simna tomorrow (call 9535145489 to confirm)

Returned to lab, and immediately transformed their comp cells using comp cell test kit (for positive control). Used plates with IPTG (100 uL of 40 mM filter-sterilized IPTG spread over and dried). Right now, going to transform our comp cells (in duplicates) using the comp cell test kit.

19/6/17

Lab notebook has not been updated in quite a while again. We should have… Anyway, stuff last week failed. One miniprep+gel went well. Some transformants of J04450 were red while others on the same plate were not. Some of the red colonies were - “papillae,” apparently. We tried miniprepping them and got a good gel only once. WT DH5A and Pichia X33 plates were streaked.

Then, the transformation work started. 3*50 uL aliquots each of TSS- and CaCl2-competent cells were transformed with 10 pg/uL, 50 pg/uL and 100 pg/uL of J04450. From each transformant tube, three plates were made: 100 uL directly, 20 uL (with 80 uL PBS) and the remainder (pellet resuspended in 100 uL PBS). Spread plating was then done carefully.

22/6/17

A test PCR was done today. The results were dissapointing as the band obtained was very weak, large an\mount of primer dimers too. Thus proper selectio0n of primers will be done and PCR shall be repeated tomorrow.

Thigns learned at Lupin

Look up stripping and redressing gas vesicles from P. rubescens and see if it has been done. Look up the protocols if so. Warning: P. rubescens toxicity (hepatotoxic microcystins). If not, think about using other gas vesicles... haloarchaeal/Anabaena?

Obtain P. rubescens (order from reputable online sources or obtain from nearby institutes). Look up protocols to culture and lyse the cells. Find someone who works on this to help us. Contact Walsby. Look at how gas vesicles are purified from them. Purify large numbers of gas vesicles, concentrate them, and store for future use. Assay their critical pressure, try centrifugation at various rpms, take images, try FACS (?), try SEM/TEM.
Try overexpressing GvpA in E. coli. Decide on a promoter and RBS, and create an assembly plan for the GvpA plasmid. Order the necessary primers, assemble the plasmid. Transform into E. coli. Three possibilities: 1) nothing happens, 2) inclusion bodies are formed, 3) entire gas vesicles are formed. Look up a way to distinguish between these possibilities. If 1), give up. If 2), try denaturing, renaturing and see if gas vesicles are formed. Otherwise give up. 3) Lyse the cells, purify large numbers of gas vesicles, concentrate them, and store for future use.
Create an assembly plan for the GvpC-linker-DDDDK-sfGFP construct that can be used in both E. coli and Pichia by adding necessary promoters, RBSes, terminators and restriction sites using primers. Order the necessary primers for everything, assemble the plasmids. Transform into E. coli and Pichia. Overexpress the fusion protein. Create a rough protein extract.
Demonstrate iFLOAT by mixing gas vesicles purified in Steps 1/2 with the rough protein extract of Step 3 and performing serial purifications. At each stage, assay the quantity of protein purified. Perform protein purity assays (HPLC).
Design an adaptor BioBrick for any protein someone might like to purify using the iFLOAT method. Create an assembly plan and execute it.

Fancy stuff we can try if we have time: Try out acoustic sedimentation of cell lysate. Look into using gas vesicles for purifying other things that have protein binding domains (GvpC-BINDER) e.g. mammalian cells [a more general purification strategy]. Look into using it for rough enzyme industries. Try optimizing process with respect to temperature, reactor topography. Try designing a continuous flow device to execute iFLOAT, say with immobilized enterokinase beads in the final chamber. Think about reusing GvpA multiple times.

01/9/17

ORders for our biobricks placed, incase more problems ovvur with minipreps and other protocols.

19/9/2017

Prepared

500 ml LB
400+200 ml LB Agar
32 petri plates
2x LB plates

Inoculated

DH5a in ml SOB (TSS comp cell prep)

20/9/17

1:30 AM

Set everything into autoclave

3:00 AM

Took everything out of the autoclave
Plates dried for 1 hr, agar stored in oven

4:00 AM

Plates dried in hood for 1 hr

5:00 AM to 7:00 AM

Plates poured
Cmp stock conc = 35 mg/ml, working conc = 35 ug/ml

21:30

TSS Protocol for comp cell prep

22:10

Set secondary culture

21/9/17

Optimizing PCR (set 1)

Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
PCR reaction mixture
- FP – 4ul
- RP – 4ul
- Stock template – 8 ul
- 2x MM – 100 ul
- ddH2O – 84 ul
Template stock – 100 ng/ul
Ran PCR 9:30 PM
Ran products on 1% agarose TAE gel (22/9, 12:20 AM)

22/9/17

Optimizing PCR (set 2)

Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
PCR reaction mixture
- FP – 4ul
- RP – 4ul
- Stock template – 8 ul
- 2x MM – 100 ul
- ddH2O – 84 ul
Template stock – 100 ng/ul
Ran PCR (23/9, 12 AM)
Ran products on 1% agarose SB gel (23/9, 11 PM)

23/9/17

Pooling PCR (set 2)

Sai - PCR 4, Sharath PCR 3
Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
PCR reaction mixture
- FP – 20ul
- RP – 20ul
- Stock template – 40 ul
- 2x MM – 500 ul
- ddH2O – 420 ul
Template stock – 100 ng/ul
Ran PCR (24/9 , 2:00 AM) and pooled products
Ran products on 1% agarose SB gel (24/9, 4:00 AM)

24/9/17

Pooling PCR 1

Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
PCR reaction mixture
- FP – 20ul
- RP – 20ul
- Stock template – 40 ul
- 2x MM – 500 ul
- ddH2O – 420 ul
Template stock – 100 ng/ul
Ran PCR (24/9 , 20:00) and pooled products
Ran products on 1% agarose SB gel (25/9, 00:30)

26/9/17

Optimizing PCR 2

Made reaction mixture of 160 ul per template , for splitting into 8 * 20 ul reactions
PCR reaction mixture (Q5)
- FP – 3.2ul
- RP – 3.2ul
- Stock template – 6.4 ul
- 2x MM – 80 ul
- ddH2O – 67.2 ul
PCR reaction mixture (Phu)
- FP – 8ul
- RP – 8ul
- dNTP - 3.2ul
- Stock template – 0.4 ul
- Buffer - 32 ul
- Phu pol - 1.6 ul
- ddH2O – 106.8 ul
Template stock – 100 ng/ul
Ran PCR (26/9, 11:50 PM)
Ran products on 1% agarose SB gel (27/9, 3 AM)

Interlab Day 1:-

Prepare 8 plates of Cmp LB agar plates
Plate the following
- Positive control –B
- Negative control – D
- Device 1 – F
- Device 2 – H
- Device 3 – J
- Device 4 – L
- Device 5 – N
- Device 6 - P

27/9/17

Pooling PCR 2

Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
PCR reaction mixture (Phu)
- FP – 50ul
- RP – 50ul
- dNTP - 20ul
- Stock template – 2.5 ul
- Buffer - 200 ul
- Phu pol - 10 ul
- ddH2O – 667.5 ul
Template stock – 100 ng/ul
Ran PCR ( 27/9, 9:30 AM)
Products were pooled (27/9 , 15:00)
No DNA pellet formed after purification and centrifugation. Thus PCR Failed

Interlab Day 2 protocols were carried out

Autoclave falcons + 500 LB broth
From each of the 8 plates , inoculate 2 colonies into 5 ml LB + cmp solutions
Incubate overnight

28/9/17

Pooling PCR 2 (attempt 2)

Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
PCR reaction mixture (Phu)
- FP – 50ul
- RP – 50ul
- dNTP - 20ul
- Stock template – 2 ul
- Buffer - 200 ul
- Phu pol - 10 ul
- ddH2O – 668 ul
Template stock – 100 ng/ul
Ran PCR at 7 PM
Ran PCR gel (1% agarose, SB) at 9:30 PM

Interlab day 3 protocol was carried out

Prepare 16 falcons with 12 ml LB+cmp media
Take OD600 of each of the above cultures. Calculate the volume required to make the OD600 of the 12 ml culture 0.02 (use the Interlab spreadsheet for that)
Aliquot required volumes of each culture into the falcons. Label properly.
At t=0 (1130), take out 500 ul from each culture and store on ice.
Incubate and shake the cultures at 37 degrees.
At t=2 (1330) , t=4 (1530) and t=6 (1730) aliquot 500 ul from the cutltures. Store on ice.

29/9/17

Optimizing PCR 5

Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
PCR reaction mixture (Q5)
- FP – 4ul
- RP – 4ul
- Stock template – 80 ul
- 2x MM - 100ul
- ddH2O – 12 ul
Template stock – 100 ng/ul
Ran PCR 6:10 PM
Ran products on 1.2% agarose TAE gel 8:20 PM (all temp gave bands)

Optimizing PCR 6

Made reaction mixture of 160 ul per template , for splitting into 8 * 20 ul reactions
PCR reaction mixture (Phu)
- FP – 8ul
- RP5 – 7ul
- RP6 - 1ul
- dNTP - 3.2ul
- Stock template – 0.4 ul
- Buffer - 32 ul
- Phu pol - 1.6 ul
- ddH2O – 106.8 ul
Template stock – 100 ng/ul
Ran PCR 6
Ran products on 1% agarose SB gel

Interlab Day 4 protocol was carried out

Transfer the collected aliquots into a 96 well plates
Take OD600 and fluorescence at 501 (excitation) and 511 (emission)

2/10/17