Team:British Columbia/Notebook

From 2012.igem.org

Revision as of 18:53, 23 June 2012

June 8

Tried to make more EPI300 cells, though no obvious growth even after hours for the second time, and decided to leave it over night to see if there are any changes.

Electrophoresed K12 with PIJ790 plasmid, and plated them on Chlor plates with control plates to examine the plates.

- Ruichen

Prepared a 96 well plate for co-culture experiments with the the following ratios:

All cells were derived from EPI300, and were grown in M9 media to allow for accurate OD measurements. a total of 10 uL culture in each case was added to 190 uL M9 media.

However, the plate reader was later found to be broken. The plate was placed in the 4° cold room until such time that the experiment could be done.

The wells were plated by GFP first, then RFP, the YFP. There was a slight accident that involved losing some of the YFP culture, but enough was recovered to fill the plates. It is likely that the GFP cultures spent over 30 minutes in the plate before the YFP was added, so some readings may not accurately reflect the ratios shown above.

-Jacob

Miniprepped biobricks 12O, 5P, 17F, 13M, 14N, 14E using the QIAGEN Spin Miniprep Kit provided by the Hallam lab. We had planned to miniprep 7B also, but a slight accident caused there to be less than 1.5 mL in the microcentrifuge tube. It will be done sometime later, after regrowing a culture

-grace

June 9

The EPI300 did grow! though over grew, and was discarded.

- Ruichen

Bought $1 of 91 octane (no ethanol) gas from the Shell on 10th. It's in a red jerrycan in the flammables cabinet near our bench.

JohnHenry 14:31, 9 June 2012 (CDT)

Experiment plan for week of June 10-16th:

1) Kill Switch Assay for Colicin E7, Colicin E2, BamH1, H₂O₂

Colicin E7: There is currently a plate of K12 cells with protein immunity and Colicin E7. Lysis protein (cuts the immunity gene to allow for Colicin E7 production) will be transformed into K12 (Sat). Cultures will be set up for both the protein immunity+Colicin E7 and the Lysis (Sun). The two cultures will be mixed and grown up to correct OD then plated (Mon). Plates will be checked for growth (Tues).

Colicin E2, BamH1, H₂O₂: There are currently plates of K12 cells with Colicin E2 and H₂O₂s. Colonies will be picked and cultures will be set up for each of these (Sat). The cultures will be grown to correct OD and induced then plated (Sun). Colicin E2 is induced low concentration of ampicillin, BamH1 is induced with arabinose, and H₂O₂ is induced with 2nM of AHL. Plates will be checked for growth (Mon). BamH1 will be transformed into K12 cells and plated (Sat). Same steps will be taken as above and plates will be checked for growth (Tues).

2) Solvent Tolerance Assay

Cultures of K12 cells with 3I plasmid will be set up (9am on Sun) in gasoline and pentane. OD will be checked every hour for 12 hours or less until stable phase (Sun). Protocol is on the TU Delft 2010 page [1].

3) PCR RFP, YFP, GFP on to pcc1 fosmid

There will be a PCR tutorial on Thursday 6pm with the primers for putting the RFP, YFP, and GFP from the biobrick on to pcc1 fosmids.

- Marianne

Wanted to start the solvent tolerance assay today, but previous protocols required 12 hours and by the time we got the gasoline, it was already 12. We decided to wait a day. In the mean time, we started some cultures and plates for a few kill switches that could also be assayed. The cultures were of a H2O2 producing strain, and another strain that expressed the colicin E2 operon, which is inducible under low (10 uG/mL) concentrations of B-lactam antibiotics, such as ampicillin (http://www.sciencemag.org/content/305/5690/1629.full#F2). Despite documentation of plating of the BAMHI kil switch, the plates were unable to be found, so another transformation was done, along with a transformation for the lysis gene from the colicin E7 operon. The colicin E7 toxin and immunity protein have already been grown in cultures. The tentative plan is to see if the two cultures interact by the lysis gene cleaving the E7/immunity complex, causing cell death. There was no one who knew how to use the plate reader in the lab today, so the co-culture experiments were further put on hold. It should be noted that the kill switches, unless otherwise noted, have been transformed into K12 E. coli cells, which are RecA positive, allowing the SOS promoter to work.

- Jacob

June 11

Autoclaved 4 500ml LB, bottle of 10% glycerol and 1 bottle of water.

-Ruichen

Started kill switch assays on colicin E2 and BamHI. Colicin E2 is on the SOS promoter, which is best switched on by mitomycin. However, we currently do not have access to mitomycin or any of its closely related derivatives, so, judging by the previous;y mentioned paper, it might be able to be induced by b-lactam antibiotics in low concentrations.

Procedure outline: Dilute 1:100 22A, 3C and wt strains in 25 mL LB. Measure OD every hour. At start of exponential phase, add arabinose to 0.01% or amp to 4 ug/mL to strain+control. Plate 25 uL 2 hours later, mid exponential phase. Details to follow.

The plate reader is still broken, with no estimated repair date.

-Jacob

Transformed biobricks 7C, 22C, and 20K into K12, 8G into BL21 pIJ790, 7C into EPI300 pIJ790, and 14C into DH5α pIJ790 for miniprep. After recovery, the cells were plated in the following manner: 22C and 20K on LB + Kan, 14C and 8G on LB + Amp, and 7C (both the K12 and EPI300 cells) on LB + Chlor. Different cells were chosen to test out the newly made competent cells. Only the K12 cells have been verified. 7C was transformed into K12 after, in addition to EPI300 pIJ790, because the 7C biobrick has Chlor resistance, which mimics the marker on pIJ790.

Quantified previously miniprepped biobricks using the nanodrop spectrophotometer.

Tested the mini-prepped plasmid concentration (using water as water)

3-13M: 43.1 ng/µl

1-5P: 111.4ng/µl

5-12O: 67.2ng/µl

7B: 70.2ng/µl

2-17F: 25.1ng/µl

3-14E: 84.9 ng/µl (260/280:1.82, 260/230:2.13)

2-14N: 223.8ng/µl (260/280: 1.83, 260/230: 2.21)

-Ruichen

- Tingchiawong

June 12

Checked plates from Jacob's June 11 kill switch assay. Every single plate (3C+, 3C-, 22A+, and 22A- in both 5 uL and 10 uL plating quantities) grew a lawn. Only 3C+ and 3C- plates had a few distinguishable colonies.

Checked plates of the cells transformed on June 11 by Ting-Chia and started 3 mL cultures for miniprepping. Only the DH5α pIJ790 and EPI300 pIJ790 transformed with the 14C and 7C biobricks, respectively, did not seem show any growth upon the first check. It was unclear as to whether there were actually any 7C (EPI300 pIJ790) colonies growing, so both plates were returned to the 37ºC room to incubate. For the other plates that did show growth, single colonies were picked and inoculated in 3 mL starter cultures for miniprep.

QIAprep Miniprep kit from Qiagen was used to miniprep 4-3I, 2-3C, and 3-22A. The cells used were previously inoculated cultures that were stored at 4ºC. DNA product was stored in sterile dH2O and frozen in the -20ºC freezer.

We received plates of Rhodococcus sp. strain JVH1, Rhodococcus rhodochrous IGTS8 (ATCC 53968), and Baillus sphaericus IGTS9 (ATCC 53969) from Dr. Jonathan Van Hamme of Thompson Rivers University today. Single colonies were picked and inoculated in 3 mL LB at 37ºC at first. Plates were then parafilm-sealed and stored at 4ºC.

After 20 hours at 37ºC, the plates with 14C and 7C (EPI300 pIJ790) had colonies. 14C only had one colony, while 7C had a couple of small colonies. The origins of the colonies were uncertain, so the plates were discarded and the cells will be transformed in K12 the next day. Also, 13M biobrick (RFP gene), which has been verified, will be transformed into EPI300 pIJ790 and DH5α IJ790 to check if the competent cells are working properly or if they are to blame for the failed 14C and 7C plates.

Acquired trp, tyr, met, and arg for use in our co-culture experiments.

- Tingchiawong

June 13

Removed 3 mL overnight cultures (20K, 7C, 8G, and 22C) from the 37ºC room and stored them at 4ºC until they are miniprepped.

- Tingchiawong

Discovered a thing Joe is too cool to do:

Update the wiki.

- JohnHenry

I approve the above message.

-Jacob

June 14

Transformed the remaining interesting kill switches without promoters into K12. Also transformed 17F into EPI300 pIJ790 and DH5α pIJ790 cells to determine if previously failed plates were due to ineffective competent cells. All transformed cultures were then plated onto appropriate antibiotic plates (+Amp or +Kan), along with a control for each. An extra control plate was spread onto a Chlor plate from a batch that previously produced unclear results. The plates were placed at 37ºC at noon and left them to grow overnight.

Made fresh batch of LB + Amp plates, which were used for half the Amp resistant samples from above. Check control plate tomorrow for colonies to ensure plates work.

Grew two 250 mL cultures of EPI300 to make into competent cells. Iced them when OD₆₀₀ reached 0.34 and 0.30. Finished glycerol stocks were stored in new freezer box "iGEM 2012 Comp Cells."

- Tingchiawong

Grace mini-prepped:

• 20K (in K12)
• 7C (in K12)
• 8G (in K12)
• 22C (in BL21 w/ plasmid pIJ790)

Ting:More EPI300 competent cells made. Need to verify competency tomorrow.

- JohnHenry

PCR Party held in the evening to start PCR experiments. PCR programmed to run overnight at keep products chilled at 4ºC until tomorrow, when gels will be ran. - Tingchiawong

Ruichen's Protocols learned for PCR:

1. get PCR tubes

2.get all reagents (except for pfu)

3. make master mix, in centrifuge tube

4. aliquot into PCR tubes (Master Mix)

5. add into the remaining reagents, except the pfu (normally jus the enzyme, and when colony PCR, pick a colony in stead of adding DNA).

6. keep sterile, pick colony (using tips, 10µl), add into tubes

7. add pfu

8. set up reaction conditions

- Ruichen

June 15

A very busy day!

1)Made 1 liter's worth of Kan plates, and about 3/4 liter's worth of chlor plates. We are now out of empty plates, and they have been placed on the list of things to buy. The Hallam lab is also out of empty plates. This is not good, for many of our knockouts are replaced by tetracycline resistance, and we have no tetracycline plates. We may have to scrounge up some from other labs. Noting that the kan plates that we used previously often grew colonies for negative controls, we increased the amount of kan stock we added from 1 mL to 1.5 mL. We have yet to test whether that is too great for the cells with the appropriate plasmid, but we have confirmed that it is lethal to normal cells by the fact that nothing grew on the negative test for the epi300 cells that Ting recently made.

2)Tested epi300 cells by transforming with the rfp cassette for easy identification of whether or not the transformation was actually successful, or if other factors were in play regarding resistance. Negative controls were also employed for the newly made plates.

• Update: many colonies on the rfp plate, but none on the negative controls. The transformation efficiency appears to be slightly lower than previous competent cells, but is still fairly high.

3)Miniprepped the resistance cassette colonies. Previously, to do the PCR, we just picked a colony, but we thought that it would be beneficial to have a plasmid stock in case we needed to repeat the PCR.

4)PCR with new (biobrick) primers was attempted. We received primers for the Dsz genes and also for the amino acid genes. We did not, however, receive the DszA forward primer, and decided to hold off on working with the Dsz genes until later. We performed a cPCR for all the genes we had, and ran the results on the gel.

5) We attempted to do a solvent tolerance assay, but were thwarted. As it turns out, gasoline has some rather unpleasant volatile compounds that were interpreted by other members of the LSC as dangerous. Also, the cells that we wanted to grow showed no growth for the first 5 hours. Personally, I think that while it would be nice to have the relevant data, it seems that the hurdles to test to see if the addition of the prefoldin chaperone helps our cultures grow in organic solvents to remove the sulfur compounds are too great. We may revisit the project, but right now, it seems that we should put the idea behind us. As of 2:18, June 16th, nothing is growing in John's other experiment, which may or not be documented on the wiki.

6)Ran the gel. Below are the results from yesterday's and today's PCR. Cameron made the gel and taped the edges of the gel box to prevent the gel from escaping. The gel itself was made to 1% agarose in 0.5% TBE, and run on a 50 well gel box. 3 uL of loading buffer were used for 8 uL (10 uL for yesterday's products) sample to increase contrast, and various ladders were tested. The negative control for today's PCR popped open during the reaction, and evaporated. Despite this, I feel fairly confident that the data is accurate.

Running a gel

1. Broad Range Ladder
2. TrpA (biobricks)+DMSO
3. TrpA
4. TrpB +DMSO
5. TrpB
6. ArgC +DMSO
7. ArgC
8. ArgE +DMSO
9. ArgE
10. MetA +DMSO
11. MetA
12. TyrA +DMSO
13. TyrA
14. empty
15. 1kb ladder
16. Mehul's Stuff metA p1002 Kan
17. Mehul's Stuff metA p1002 Kan
18. Mehul's Stuff metA p1002 Kan
19. Mehul's Stuff metA p1002 Kan
20. empty
21. Ruichen's Stuff
22. Ruichen's Stuff
23. Ruichen's Stuff
24. Ruichen's Stuff
25. Ruichen's Stuff
26. empty
27. 1kb + Ladder
28. TrpA tet
29. TrpA tet +DMSO
30. TrpA tet +MgCl2
31. TrpA tet +DMSO+MgCl2
32. ArgE tet
33. ArgE tet +DMSO
34. ArgE tet +MgCl2
35. ArgE tet +DMSO+MgCl2
36. TyrA tet
37. TyrA tet +DMSO
38. TyrA tet +MgCl2
39. TyrA tet +DMSO+MgCl2

As you can see, we have successful bands for tyrA (from genome, biobrick primers) with DMSO, MetA with kanamycin resistance (for knockouts), trpA with tetracycline resistance, ArgE with tetracycline resistance, and tyrA with tetracyline resistance. The 1 kb+ ladder appears the most useful in this experiment.

- Jacob Toth

Jacob summarized well, and I am just going to add more info based on his summary.

As for the perish dish, we used up all ours and also used the remaining a bag and half of Hallam Lab's. (which we need to reimburse back to them).

I (Ruichen) added perish dish (10 bags) to the Hallam's Lab ordering list, using iGEM's name.

As for the organic solvent tolerance assay, indeed there has been no growing sign in the transformed culture (with 3I plasmid) for the first few hours, and it was left in the 37 degree room over night.

and for the controlled cultures, they were growing as expected when the organic solvents (Pentane and Octane) were not introduced, the experiment was continued (with the disruption of Jacob mentioned before). The organic solvent were added into controlled cultures at different concentrations (0%, 5%, 10%, and 15%), and their growth were monitored roughly each hour for the first few hours, and when the disruption occurred the cultures were taken out of the 37 degree room and stored in the 4 degree room at 5 p.m.

I (Ruichen) returned to the Lab after dinner at 8:30 p.m., and each culture were examined for OD readings again, and the cultures with Pentane and Octane were incubated in the incubator with vacuums at 37 degree for 1 hour and 30 minutes respectively. Then the OD were read again at 9:30 p.m..

The detailed OD reading data will be posted later with analyzed results.

- Ruichen

The Amp plates made the day before worked. However, the Kan plates (negative control, EPI300 pIJ790, and DH5a pIJ790) showed unexpected results. The negative control, which only had untransformed K12 cells plated, had more colonies growing than those from the plates with resistance-transformed EPI300 pIJ790 and DH5a pIJ790. Even then, there were only a few small colonies on the EPI300 pIJ790 plate and none at all on the DH5a pIJ790 plate. It looks like the Kan plates may not be working, and it is possible that the EPI300 and DH5a cells with the pIJ790 plasmid are not competent.

- Tingchiawong

June 16

The time I came in Joe was making the PCR gel.

The cultures with 3I plasmid that left for growth in the 37 degree room actually did turn a bit cloudy (6:00 p.m.).

Their ODs were then measured with a blank LB for reference, and the detailed info will be updated.

For the remaining cultures, 100µl of each culture was tested on Chlo plates with an control of K12 (20µl) to see if they are actually the cell cultures that of our interest, and also testing the chlo plates that are newly made, and the remaining cultures were stored in the 4 degree room for potential future usage.

The plates were left in the 37 degree room, and will be collected by Joe and stored in the 4 degree room.

- Ruichen

June 17

As shown above in the gel image, there is still some PCR that needs to be done. Additionally, it appears that Joe's knockout PCR was not working either, so that PCR was to be repeated as well. It appears apparent that increased concentrations of DMSO and magnesium chloride help the reaction, so the appropriate amount of each was added to the mastermix. The reactions were as follows: TrpB with Kan cassette, TrpA, TrpB, ArgC, and MetA.

Since we were planning to put these into biobricks, it was imperative that we made sure that there were no restriction sites. To do this, we used the NCBI data for the gene sequence and ran it through Nebcutter, searching for all illegal sites. No illegal sites were found in TrpB, TyrA, TrpA, ArgC, or MetA. Lucky.

As shown above, a reaction for the tyrA gene worked. Using this PCR product and the rfp plasmid, we digested with EcoRI and PstI and ligated with T4 DNA ligase. The resultant product was used to transform Epi300 cells. Tomorrow, we will check for white colonies among the red ones, which would suggest that the ligation was successful. A colony PCR will be done to confirm.

-Jacob

June 18

Yesterday's plates did not grow, but the experiment was repeated today.

1) Made tetracycline plates using LB agar and Hallam lab tetracycline, diluted 400 uL in 400 mL. Previous kan and amp plates were shown to not have LB in them, and were also discovered not to support growth. However, a working kan plate was gifted by Cameron and used for a knockout experiment.

2) A gel from yesterday's PCR was shown to have some products that worked, while others did not. The MetA, TrpA, TrpB, and ArgE biobrick PCR's worked fine, but the ArgE-Kan cassette and the ArgC biobrick PCRs did not, and were not repeated today. From personal correspondence with Joe, his PCR was not working either, despite trying different cycles and conditions. He suspects that there may be something wrong with the template, given the inconsistent performance of the Kan plates.

3) The biobrick PCRs that did work were digested with EcoRI and PstI, as was the psb1C3 linearized plasmid backbone. it was unknown whether or not the plasmid had any methylation, so DpnI was used only in the PCR product digestion. The procedure will be uploaded to the wiki in the near future. A gel was made showing the ligation products.

4) The biobrick ligation products were used to transform K12 cells and were plated appropriately. The successful amino acid-antibiotic cassettes were also transformed into EPI300, DH5a, and BL21 cells with the appropriate recombineering plasmid, and plated. To date, all the tet resistance strains have been plated, as well as Mehul's Amp resistant strain. The kanamycin resistant strains remain recalcitrant, perhaps due to, again, the inconsistent performance of the kanamycin plates.

5) Joe, looking into rhodococcus growth, decided to make some TB.

It should be noted that several of the transformations sparked during the electroporation procedure. Some success has been reported with sparked cultures, so they were plated anyways.

As an update, here is a table showing what has been done so far.

It should be noted that when a ligation has been done, it does not mean that it was successful. We will not know until we see growth on the plates and a cPCR of the colonies has been done. Even then, it would be useful to have it sequenced.

-Jacob

Worked with Jacob on steps 3 and 4 listed above. I made the gel, practised loading samples into wells (with Ruichen), and learned that the machine used to see the results is finnicky. Unfortunately, the 1 kb reference ladder did not work (unexpectedly and for unknown reasons).

(Step 3) Transformation and plating of the biobrick PCR products were the last things we did today. Of the 5, only TrpB sparked during electroporation. 1 uL of PCR product was used to transform cells.

(Step 4) Of the successful amino acid/antibiotic cassettes, Jacob and I transformed genes "4", "7", "12" (Tet resistant) and "M3" (Amp resistant)- names of the genes to follow - into EPI300, DH5a, and BL21 all with the recombineering plasmid pIJ790. The ones that sparked during electroporation are: 4 (EPI300), 4 (DH5a), 7 (BL21), and M3 (BL21). Quite a mix, but the addition of DMSO and MgCl₂ to boost PCR efficiency increased the salt concentration of the solution and may have caused sparking. Gene 4 had both compounds added and 7 had DMSO (unsure about M3 - that was Mehul). Only 1 uL of PCR product was used to transform cells to reduce chances of sparking, which may have helped.

- Grace.yi

June 19th

Designed primers with Jacob and Mehul

- Ruichen

Designed primers with Ruichen and Mehul.

More specifically, the primers were for constitutively expressed GFP, RFP, and YFP to be put in the PCC1FOS vector. An EcoRI site was added just before the start of the promoter, and a BamHI site was added to the 5' end of the reverse complement. The melting temperature was normalized to about 59°C, and the primers were checked for any secondary structure.

There were no colonies on the plates transformed with any putative biobricks, so the procedure was attempted again.

-Jacob

June 20th

Plated the cultures with 3I plasmid on Kan for control and Chlor for testing purpose.

- Ruichen

Still no biobrick colonies. Tried again with an altered ligation protocol. Also ordered a variety of kill switches from the registry, along with Joe's Panamanian Pseudomonas Rhamnosyltransferase. Repeated PCR on things that had previously failed, and got the final amino acid biobrick and Ruichen's knockout resistance construct. Joe's knockout resistance cassette remains recalcitrant. It should be noted that I ran reactions with either PFU or phusion, and only the phusion worked. This may have been because of the low volume (0.5 uL) used.

At the meeting, the gasoline debacle was discussed, as was the general progress of the project, and potential fixes for some of the problems that we have been having.

I suggested to Joe that he use the 2-17F biobrick for his forward kanamycin resistance cassette, and other ideas were thrown around as well.

-Jacob

June 21st

The culture has been proven to be transformed successfully with the 3I plasmid! They grew colonies on the Chlor plate and not the Kan plate.

- Ruichen

Today was relatively fruitful. All amino acids have been PCR'd out of the genome, all the resistance cassettes are ready for the knockouts, and several colonies appeared on the ArgE plate. One colony was selected and grown in culture. The rest of the biobrick ligations were redone and allowed to ligate overnight.

The digests of the products were done with EcoRI and PstI in buffer 3, as opposed to the EcoRI-HF and PstI in buffer 2 we did previously. This was done because PstI has 100% activity in buffer 3, as opposed to 75% activity in buffer 2. Also, we learned that EcoRI-HF has 0% activity in buffer 3.

-Jacob

June 22nd

Transformed with the overnight (Room temperature) ligation, using 1 uL ligation mix and 1800V. Joe then plated the cells after recovery.

Cameron reported knockouts!

Found a MetA biobrick colony on the appropriate plate from 2 days ago. Decided to grow it up in culture and run a PCR to confirm that it was successful.

Had previously grown up 10 mL of each fluorescent strain (GFP, RFP, YFP), but the plate reader became unavailable.

Wanted to design mutagenesis primers, but couldn't remember what software to use. For future reference:

NCBI for FASTA

Nebcutter to find sites

Virtual Ribosome for associated peptide

Wikipedia for codon table

PrimerX for basic design.

To whom it may concern, I think doing 4 sequential site directed mutagenesis reactions on dszA is going to take far more time and nearly as much money as simply synthesizing it.

Joe appears to be successfully growing IGTS8 (Rhodococcus) and IGTS9 (Bacillus) in TB at 30°C, and Pseudomonas putida at 37°. He also reports being unsuccessful in doing a cPCR for the dszD gene.

-Jacob