NYU Gallatin 2012 iGEM Team
I used the purified DNA pieces from 8.22.12 that Steven and Min purified (they’ve been at -20C so they should be okay).
Alkaline phosphatase the A-backbone:
10 uL DNA
3’ overhangs require 15 min incubation at 37C (5’ overhangs require 60 min). SpeI leaves 3’ overhangs. Incubate 15 min at 37C...actually 45 min because the water bath isn’t up to inactivation temp.
2 uL A-pSC1C3
Also set up one ligation without insert (no N). Make up the volume with water so total is still 10 uL.
Incubate 1 h at RT.
Transform by heat shock transformation protocol into commercially competent DH5alpha cells. 2 transformation: one with 2 ul ligation, one with 8 ul ligation. Plate on LB-chlor plates. Incubate at 37C overnight.
During this time, we purified the DNA digested yesterday. We will ligate tomorrow using this DNA if today’s transformation is unsuccessful.
The following PCR conditions did not work:
Reducing the primer:template ratio 10x
More NAG1 and UAP1 DNA inserts were liberated with SpeI and PstI. Plasmid containing A was linearized with SpeI.
The correct pieces of DNA (circled) were cut out and put in the freezer, to be purified later
We filled 2 more molds, 10R and unknown. Our biggest problem so far is cracks and holes; its unknown the best way to do it, as the tape might be the reason for contamination. Temp: 29.5
Restriction Enzyme Digest #2 (re-run):
Reaction ran for 30+ minutes.
2ul of 10x Bromophenol Blue loading dye
Same results as yesterday.
Made 4ml cultures of new colonies: clones #16-22 are in the incubator at 11:40pm along with a neg. growth control tube..
PCR with A gene is running.
No growth in colonies 3, 4, 7, and negative control for growth.
Restriction Digest (12 plasmid preps total) with EcoR1 and Pst1 was run at 37*C at 9:45pm for 30 minutes.
Master Cocktail (13):
10ul of cocktail mix and 10ul of DNA were combined.
RE digest products were run on a 1% gel for 40 minutes to see if an insert of 4kb comes out of the 2kb pSB1C3:
20ul DNA digest product
Lane 1: 10ul 1kb PLUS ladder
Cannot see 4kb insert in any lanes, and can see 2kb backbone in lanes 13, 14, 15.
Two more molds were contaminated, but there are solid growths on both the tubes and most molds. We filled four but unfortunately, we ran out of substrates and the autoclave was malfunctioning. Temp in incubator lowered to 29C
Both plates showed colony growth, with Gibson Construct plate #1 (higher dilution) showing more growth than Gibson Construct plate #2 (lower dilution).
Made Chlor LB broth media:
Picked 15 colonies and grew overnight in 4ml culture tubes
Transformed E. coli with Gibson construct were plated onto two plates (see Ellen re: dilution)
PCR Purification (A, N, U)
· Add 4 volumes of PureLink Binding Buffer (B2) with isopropanol to 1 volume of the PCR product (50–100 μL). Mix well.
Made 1% Gel
· Ran in a gel for ~15-20 min
Cutting the gel and purifying
· Equilibrate a water bath 50 C
Purification procedure using centrifugation
· Load the dissolved gel mixture with DNA onto the center of a pure link wash tube
Made 1% Gel for PCR Purification for plasmid prep a4
Very little yield for linearized AGM1 plasmid but it is there. Std was 5uL, all others 10uL per lane. The smaller. The frag the more efficient the purification.
Transformation (Gibbson assembly)
Version 1 Version 2
7ul AGM1 plasmid 9ul AGM1 plasmid
20ul total 20ul total
*PCR (version 1 and version 2) is in PCR machine
I filled two molds and was unable to fill more since the autoclave was being used and it could not wait to autoclave more. Incubator temp 31
The lab strains look the same. Ph same as well as temp
Lab strain looks much better and uniform from last time, a sheet is expected. Ph 4. 5
The 10 beakers were moved to the incubator, temp. 30. Two more molds were. Contaminated and will be filled in. In all we filled 4 molds and 11 small tubes for test samples.
Kombucha-temp is 23. 5 in cabinet.
Lab strain in blueberry juice is contaminated. Ph 2
Wild kombucha is growing best but still weakly. Ph 4. 5
We moved the molds out of the incubator and into cabinets in the lab. Four from before were contaminated, they were cleaned and four more molds were made. 5 star shapes were also put together.
Came into lab. Jimmy had already made the substrate, we filled out 5 of the 9 molds needed with the oak pellet substrate. To avoid contamination, we wrapped the finished molds in plastic wrap. We also used aluminum star shaped containers, in order to create smaller molds. Two and a half. All molds were made with h2o2 mycelium. No new contaminants, though one seems to be developed.
Kombucha. Made three with lab strain scoby, two with aceto food, one with blueberry juice
Came into lab today, 7 out of 16 molds were contaminated. It seems like the tapes used to seal up cracks are the culprits as most of the mold growing seemed to originate at the tapes. There needs to be a better way to seal things up. what was done: we merely cleaned out the contaminated trays and are leaving the rest for tomorrow.
Ligation & Transformation
Two ligation reactions:
The second one (that is a bit of a crapshoot but could potentially put all three pieces together):
Centrifuged for 20 seconds at 2.0rcf because T4 ligase was pipetted onto the side of the tube for Lig 2.
Incubate at room temperature for 30 min to 1 h.
*** We should have used CIP/phosphatase so that the AGM1 gene with the pSB1C3 vector cannot religate... Since we did not, we are expecting to see more religated plasmids without the intended inserts for both Lig 1 and Lig 2 rnx’s.
Transformation set up for Ligation 1 and Ligation 2:
Pellet for 8.4rcf
PCR products of the N and U gene plasmid digests were retrieved and run on a 1% gel at 3:08pm for about 40 minutes.
5ul of 1KB Plus DNA ladder
2uL PCR product
Lane 1: empty
Predicted band sizes:
Also, running the gel for 40 minutes was way too long-- next time only 25-30 so our product doesn’t go off the gel!
PCR products (tubes A, B, C, D) are in the hot pink “Cloning Materials” box and labeled “Genes N/U RE & PCR Products 8/23”
We purified our PCR products via miniprep. Only then did we look more closely at the gel to see that our "products" were in the 250-400 bp range, when we expect 800-1600 bp range. Clearly the PCRs failed.
In examining the cause of PCR failure more closely, we realized that the reverse primer for gene 1 (AGM1) is incorrect. The reverse complement of the forward sequence was never generated (see the "primers" google document to confirm this). The good news is that it's easy to generate the correct sequence and order the primer. The bad news is that we can't do the final gibson assembly without it.
To troubleshoot the PCRs that should have worked (NAG1 and UAP1), we are running PCRs again overnight. We are using different concentrations of primer/template and testing different Mg2+ concentrations. This time our reactions looked like:
Forward primer (10 uM): 1 ul
Forward primer (10 uM): 1 ul
Our PCR cofactor (1% MgCl2) was:
A Tube: NAG1
B Tube: NAG1 with MgCl cofactor
C Tube: UAP1
D Tube: UAP1 with MgCl cofactor
We also decided to start cloning the classic digest-and-ligate way using our genes which are already in the biobrick plasmids. We digested AGM1 plasmid with SpeI (thus linearizing it). We digested NAG1 and UAP1 plasmids with SpeI and XbaI, liberating the genes (and RBS) from their backbone plasmids.
We ran a gel of our digests, and they look great - the exact sizes we expected (I will attach it later). Another confirmation that our minipreps contain the genes we expect them to (even if we don't have the full sequence). We cut the pieces out of the gel that were the correct sizes and gel purified them. They are in the fridge and ready to ligate together.
We ran a gel to confirm that we have PCR products of expected sizes. I glanced only quickly at the gel, saw that there were some bands (admittedly diffuse but the gel itself was a week old). The gel is attached (sad gel.png). Its name will become clear tomorrow.
We set up PCRs of the genes using primers containing overlapping sequences. For each PCR, we diluted the dessicated reagents (Taq Polymerase, dNTPs, MgCl, buffer) in the following:
Forward primer (10 uM): 2 ul
(I may be off. I didn't take notes here. If I'm wrong and you remember, please let me know.)
The DNA sequences came back. None of the forward reactions worked (especially surprising as these primers were supplied by iGEM headquarters). Of the reverse reactions, some worked and some didn't. There was no read for the NAG1 gene, for example, but as we only had one digest that looked positive, we have to move forward with this clone (for now).
The best hits were:
AGM1 - clone 4
The cultures were well grown the next day (and the original colonies were still white, a promising sign). We miniprepped the plasmids from the cultures, and then used an XbaI/SpeI digest to identify plasmids with the correct-sized inserts. The expected sizes for the DNA fragments:
1.6 kB - AGM1
The digests were run on a 1% agarose gel. I've attached the picture (gel annotated.png) that shows the inserts with the correct sized bands (the samples with the asterisks). These were sent for sequencing using the VF and VR (verify forward and verify reverse) primers.
The next day we had lots of colonies (I don't have the pictures of the plates, but maybe someone else does). Many of these were red, indicating they were transformed due to residual RFP plasmid (as had happened previously). We circled several white colonies as prospective positive transformants. For each construct, we chose 5 clones and inoculated 4 ml of LB-chlor to grow these overnight at 37 deg C.
The second gibson assemblies that we did worked! We followed Ellen's advice to increase the G-bit DNA concentration. This means our final assembly reactions looked like this:
2.5 ul pSB1C3 (biobrick backbone plasmid DNA, linearized)
* we divided the volume left by the number of G-bit parts. For NAG1, there were 3 parts, so each took up 2.5 ul. For UAP 1, there were 4 parts, so each took up 1.8 ul. For AGM1, there were 5 parts, so each took up 1.5 ul.
We incubated these for 1 hr at 50 deg C and then immediately transformed the commercial competent cells.
Gibson assembly protocol
-reaction is in 0.2mL PCR tube using program “Gibson” under user “ellen”
Results of Gibson Assembly:
Lawn on antibiotic free control
Some contamination on A & U plates
No growth on N, + Control plates
[PASTE ALL PHOTOS FROM ALEX EMAIL AFTER RESIZING]
Pink colonies may be traces of RFP plasmids remaining from when biobrick plasmid part was PCR’ed.
Reasons for failure could be:
1) use the heated lid on the PCR machine - I did not shut it because I thought it might jack up the temp.
Notes for NAG1 Gibson
Gibson Assembly Master Mix
4.3 μL water