William

PCR on BL21 and AB2848 strain on bacteria to retrieve LacZ-beta gene. Used LacZ-beta STOP primer.

Agarose gel electrophoresis result: Band about 3 kb for the BL21 strain. Probably LacZ-beta.

William

PCR clean-up using Machery-Nagel PCR clean-up kit. Followed protocol from the kit.

PCR amplification of the cleaned DNA with and without stop codon.

William

PCR clean-up using Machery-Nagel PCR clean-up kit on DNA with and without stop codon.

We did step 1 of making E. coli TOP10 cells competent (incubation of cells from freeze stock in LB medium with streptomycin.

We analyzed the DNA concentration using NanoDrop:

• LacZ-beta with stop codon: 230 ng/µl
• LacZ-beta without stop codon: 300 ng/µl

William

Finished Jack's protocol for making competent cells.

Attempted to transform bacteria with these parts:

• LacZ-alpha 22D (plate 2)
• 10X His-tag 5P (plate 3)
• pBad strong promoter 14A (plate 3)
• T1 terminator 1D (plate 1)
• Double terminator 3D (plate 1)

Parts were resuspended in accordance to iGEM protocol and for the transformation we used Jack's protocol.

Plates for E. coli growth were made and the transformation products were plated.

William

Growth of 10X his-tag and double terminator transformed bacteria observed. Overnight (O/N) cultures were made of these.

LacZ-alpha, pBad and T1 were attempted transformed again.

New batch of LB-plates were made with chloramphenicol.

William

Restriction cutting of LacZ-beta STOP cleaned PCR product, LacZ-beta NONSTOP cleaned PCR-product and pSB1C3 were attempted using restriction enzymes XbaI and PstI. Incubation in accordance with iGEM restriction digest protocol. The tube with pSB1C3 was not mixed with water before pipetting.

The concentration of the digested products were measured in NanoDrop:

• LacZ-beta stop: 14.4 ng/µl
• LacZ-beta non stop: 23.3 ng/µl
• pSB1C3: 26.5 ng/µl

Ligation was attempted on the digestions using T4 DNA ligase and following the iGEM ligation protocol.

Freeze stocks of the O/N cultures of 10X His-tag and double terminator were made.

Minipreps using Promega Wizard Plus SV miniprep kit was attempted on saturated 10X his-tag and double terminator cultures.

Cultures observed on pBad, LacZ-alpha and T1-terminator plates. O/N cultures made during the weekend.

William

No colonies seen on the LacZ-beta stop and non stop plates.

The digestion was repeated, but now with solubilized pSB1C3.

William

Made O/N culture of T1 terminator from freeze stock in order to obtain pSB1C3 plasmids in case the pSB1C3 from the tube in the kit is defective.

We tested the restriction digest protocol by using a part from the kit we don't need (1A). Due to issues with the agarose gel we started over using another part (1K) that we left it in 37C for 4 hours and then on 4C overnight.

William

Miniprep of the T1 terminator O/N culture. 3 MP's made in parallel with NanoDrop DNA concentrations:

• T1-1: 15.6 ng/µl
• T1-2: 16.7 ng/µl
• T1-3: 16.2 ng/µl

The T1-1 was digested using XbaI and PstI following the iGEM digestion protocol.

The product was run on a gel and we cut out the pSB1C3 part using a scalpel and stored it in a eppendorf tube in the fridge.

William

We used the NucleoSpin gel clean-up kit and protocol in order to isolate the XbaI/PstI-cut pSB1C3 from the gel piece. Concentration measured with NanoDrop: 10-15 ng/µl.

PCR was done on LacZ-beta with stop and non stop reverse primers using Q5 polymerase.

Vilde

Made N-Histag from primers. Used the program called “gradient” in the PCR machine. Started at 95 deegree and went slovly down to 55 degrees. Found out that this was the wrong program to use.

William

O/N cultures were made of the colonies of transformed pSB1C3/LacZ-beta non-stop.

PCR was run with VR2 and VF primers for confirmation.

Vilde

Made new N-term His-tag primers. Used Waterbath this time. Put the waterbath at 95 degrees, mixed the Forward and reverse oligoes for the N-his term and put them in the waterbath. Let the temperature slowly go down to about 50 degrees.

Vilde

Made a big O/N culture of the pSB1C3 C-term His to isolate the plasmid backbone tomorrow. Made a 50ml O/N culture with chloramphenicole. 50ul Chloranphenicole in 50ml LB medium.

William

3A assembly was attempted with the bricks pBad and 2I, 2K, 19E and 11N, using pSB1A3.

Vilde

Miniprepped lacZ-beta Non-stop pSB1c3 (Wizard Miniprep). Final Concentration of 95 ng/ul.

Vilde

Quickchange mutagenesis of LacZ beta non stop. One of the parts we are making contains an EcoRI site. We will do a mutagenesis to get read of this site. Used 7,5ul primers and 6 min elongation. Followed the quickchange protocol and used pfu Turbo enzyme from agilent technologies.

Vilde

A3 assembly of lacZ Alpha Stop and LacZ beta NS into pBAd.

Vilde

No colonies after the mutagenesis or A3 assembly. Troubleshooted it.

William

Batch of LB agar plates w/Ampicillin was made.

Vilde

Made primers for Autotransporters.

Vilde

Ordered primers for Autotransporters, Signaling peptide, Primers to amplify linearized plasmid backbone and VR, VF2 primers.

The primes contains the standard prefix and suffix. Non of these genes contains any of the restriction sites for the restriction enzymes in the iGEM assembly protocol.

• Invefull Long
• Invfull Short
• Ehaj1 – Short
• Ehaj2 – long
• Signalling peptide

Vilde

Assembled lacZ beta Stop and NS (Cleaned up PCR products) into pSB1C3 plasmid.

Cut both plasmid and parts with EcoRI and PstI. Ligated for 1h at RT. Transformed 2 ul.

William

The results of the 3A assembly was poor, with either no growth at all or carpet growth.

The O/N cultures from the day before was made without using antibiotics, so the cultures was transferred to fresh medium with antibiotics to try to select for the right bacteria.

2I, 11N, 2K and 19E was plated on LB agar with 0.1% arabinose.

New batch of LB agar was made, using LB broth powder.

Vilde

No growth observed from the assembly into pSB1C3. We think there may be something wrong with the linearized plasmid. We will try to amplify the plasmid by PCR from another part and use that one in the assembly work.

Vilde

• Found out that the iGEM scar contains a Stop codon
• Found tRNA amber Stop – part in the kit
• Found a constitutive promoter
• Found plasmid (from jack) with another origin of replication

Vilde

• Transformed Amber stop – tRNA and promoter
• Amber stop tRNA - Part: K228001, 14E kit plate 3
• constitutive promoter – Part J23119, 17O kit plate 3

Vilde

Got colonies from transformation of the amber stop tRNA and promoter. Tested the colonies on PCR with the VF and VR2 primers. Made O/N cultures in LB chloramphenicole medium.

William

O/N cultures was miniprepped and freeze stocks were made of:

• pBad + 19E in pSB1A3
• pBad + 2K in pSB1A3
• pBad + 2I in pSB1A3
• pBad + 11N in pSB1a3
• 13L in pSB1A2
• His alpha stop in psB1A3
• 19E in pSB1C3

All in TOP10 E.coli cells.

New transformation was set up using the following ligation products:

• LacZ alpha NS (from AB strain) + pBAD + pSB1A3
• LacZ alpha NS (from BL strain) + pBAD + pSB1A3
• LacZ beta NS (from AB strain) + pBAD + pSB1A3
• LacZ alpha NS (from BL strain) + pBAD + pSB1A3

The NCM17 E.coli strain was received from Yale E.coli Repository, plated and overnight cultured.

Vilde

Miniprepped and made freeze stock of Amber tRNA and constitutive promoter.

William

Freeze stock was made of NCM17 strain.

Vilde

Tested the function of the pfu Turbo enzyme. The enzyme was forgotten at RT for 48h. I therefore tested if it was still functional by running a pcr on genomic DNA with Actin primers. I used the pfuTurbo protocol 600410. Results: No bands. The PCR was repeated with different primers and substrate but still showed no results. We concluded with that the enzyme is not functional anymore.

William

O/N culture of bacteria transformed with 17O was miniprepped.

Vilde

Cleanup-day in the lab! We tested all the minipreps we got in the freezer. Used Takara enzyme and general reaction mix. Tested them all on PCR and run a gel afterwords. We found out that all our plasmids contain the gene we expected.

William

Made 20 LB plates with kanamycin.

The miniprep of LacZ beta NS in pSB1C3 was run on a gel. The bond was the right size.

Vilde

A3 assembly of constitutive promoter and amber stop tRNA. Followed protocol from iGEM kit. With some small changes – used only ¼ of the reaction volume.

• Part A: Constitutive promoter
• Part B: amber stop tRNA.
• Linearized plasmid: pSBA1A3

Since the RNA made from the amber stop tRNA gene will not be translated into proteins, a RBS in front of the gene is not necessary. I therefore ligated the promoter directly to the gene.

• Digestion buffer: NEB 3.1
• ligation time: 1h at RT.
• Transformed 3 ul of the ligated product.

William

The NCM17 strain was made competent (Using Jack Leo’s protocol) and put in freezer.

Vilde

No growth observed after the A3 assembly. Troubelshooting.

Vilde

New Assembly of amber stop tRNA and the constitutive promoter. Tried the assembly again, but this time with a larger reaction volume, 1/2 of the original volume. Also used the cutsmart buffer and not the NEB 3.1 buffer. I also changed and used the pSB1K3 plasmid instead of the pSB1A3 plasmid.

Vilde

Received primers for autotransporters and signaling peptide. Made stock solution and user stocks (5uM) of the primers.

Vilde

Growth from the A3 assembly of amber stop tRNA and constitutive promoter. Checked colonies on PCR with VR and VF2 primers and made O/N cultures of positive colonies.

Vilde, Sumaya

Minipreped and made freeze stock of Izadoras O/N cultures of LacZalpha in pSB1K3. Used wizard miniprep protocol.

Vilde

A3 assembly of His-Tags into LacZ beta. Used A3 assembly protocol from iGEM.

1. Part A: N-term His
   Part B: B-Stop
2. Part A: Beta-Non Stop
   Part B: C-term His
3. Part A: C-term his
   Part B : T1 terminator

Found out that these lacZ beta parts still contains the EcoRI site. The samples will not be used. Assembly nr.3 (C-term-his to T1 terminator) will be used in future work.

Vilde

We found out that the promoters we have been using do not contain a RBS in front of it. We will therefore need to make new constructs with a RBS in between the promoter and the gene. I found and transformed these RBS from the kit:

• RBS 4G kit plate 4 in psb1C3
• RBS 1N Kit plate 4 in pSB1A3
• RBS-LacZ alpha 22D kit plate 3 in pSB1C3

Vilde

Made O/N culture of the strain we will use to express the amber-stop tRNA. It contains CAM resistance. Place 69# in Dirks stock.

Vilde

Plated out bacteria from Dirks stock witch contains the plasmid that contains the autotransporters with the signaling peptide.

• Invfull #289
• Ehaj1 #261
• Ehaj2 #262

All these plasmids got amp resistance.

Vilde

Made constructs of Autotransporters and signaling peptide. Used the Q5 protocol and did PCR on bacteria colonies I grew up 21.07. Tested the PCR results on a gel. Got bands for every constructs at the correct length. I made: Ehaj1, Ehaj2, Inventful short, Inventful long, Signalling peptide.

Vilde

3A Assembly of autotransporters and signaling peptide.

Followed the protocol and the manual from the iGEM kit. Used pSB1K3 as linear plasmid.

1. Part A: Signaling peptide
   Part B: Inventful long
2. Part A: Signaling peptide
   Part B: Inventful short
3. Part A: Ehaj1 short
   Part B: T1 Terminator
4. Part A: Ehaj2 long
   Part B: T1 terminator

Vilde

Got Colonies from the 3A assembly of autotransporters. Made O/N cultures.

Vilde

Assembled parts into pSB1C3 – The shipping plasmid. Used linearized plasmids pSB1C3 from the kit. Cut both plasmid and parts with EcoRI and PstI. Digestion and ligation followed protocol. Ligated these constructs into pSb1C3: Ehaj1 short, Ehaj2 long, Invful long, Invful short, Signaling peptide. Transformed into E.coli. No growth observed the next day.

Vilde

Run LacZ Beta Stop and Non Stop on gel to make sure they were in a linear plasmid. No results on the PCR.

Vilde

Tested lacZ beta colonies on PCR. Used TaKaRa setup and protocol. Tested LacZbeta Stop, lacZ beta non-stop, lacz beta with N and with C-term his. Nothing was observed in the gel. Not even the ladder so we might ha forgot the DNA Dye.

Vilde

Transformed the part for galactosidase into e.coli

Vilde

Mutagenesis of lacZ beta Stop and non-stop. Used QuickChange protocol and setup with pfuTurbo enzyme and dpnI treated the sample. Used LacZBeta Non-Stop-pSBiK3 and LacZbeta Stop- PSB1K3 as template. I Transformed 3ul into bacteria. No colonies was observed the day after.

Vilde

3A assembly. Used standard protocol and procedure. Ligated them all into the psb1T3 plasmid.

1. Part A: alpha NonStop
   Part B: Ehaj1 short – T1
2. Part A: Alpha NonStop
   PartB: Ehaj2 Long – T1
3. Part A: Beta NonStop
   PartB: Ehaj1 short – T1
4. Part A: Beta NonStop
   PartB: Ehaj2 long – T1
5. Part A: Signalling peptide (SP) - Invful short
   PartB: Alpha Stop
6. Part A: SP- invful short
   Part B: Beta Stop
7. Part A: Sp-invful long
   Part B: Alpha Stop
8. Part A: Sp-invful long
   Part B: Beta Stop

Vilde

Assembled the Amber stop tRNA into plasmid. I will assemble the promoter-AmberStop tRNA into the plasmid; pACYCDuet1. I cut both plasmid and part with EcorRI and PstI. The plasmid contains a multiple cloning site that will be used to set in our part. The plasmid contains chloramphenicol resistance.

Vilde

Made O/N cultures from the 3A assembly done yesterday.

Vilde

No growth in the O/N culture except from the promoter-Amberstop-tRNA in the pAC-plasmid. We found out that we had used the work tetracycline antibiotic.

Vilde

PCR of lacZ beta from the galactosidase biobrick from iGEM kit. This galactosidase gene does not contain the EcoRI restriction enzyme in the end of the gene. This means we do not have to do mutagenesis to take away the restriction site. I use Q5 protocol and setup. Used primers: LaczBeta forward and LacZBeta reverse – Stop and Non-Stop. Used both galatosidace biobricks found in the kit as template. For the part 9E kit plate 2 (galactosidase w. RBS) I got clear bond for both parts. We will use these parts for future work.

Vilde

Made O/N culture of RBS-4G in pSB1C3. We will use this to isolate the plasmid, cut it and run it on a gel. We will isolate the plasmid from the gel and use it in the assembly.

Vilde

Meeting with supervisor about Boston/Giant jamboree and problems in the labwork.

Vilde

PCR Clean up. Cleaned up PCR products before digestion. Used NovoSpin Gel PCR and clean up kit. Washed two times and eluted in 20ul of dH2O. I cleaned this PCR products:

• Invful Long
• Invful Short
• Ehaj1
• Ehaj2
• Signaling Peptide
• LacZ beta Stop
• LacZ Beta Non Stop
• LacZ Alpha Non Stop

Checked concentrations on Nanodrop: ca 40ng/ul

Vilde

Digested samples for future 3A assembly and ligation into pSB1C3. Digested with EcoRi and SpeI:

• Promoter-RBS
• LacZBeta Stop
• LacZBeta Non Stop
• LacZAlpha Non Stop
• LacZAlpha Stop

Digested with XbaI and PstI

• SP-invfull short
• SP-Invfull long
• T1 (Terminator)
• Ehaj1-T1
• Ehaj2 – T1
• C-term his – T1

Digested with EcoRI and SpeI

• pSB1A3
• pSB1K3
• Samples I PCR cleaned up earlier today

Digestion: 30 min at 37 degrees and inactivation of the enzymes by 20 min at 80 degrees.

Vilde

PCR Clean-Up. Used Nucleospin gel and PCR clean up and cleaned up all the digested samples.

Vilde

Run gel on Sumayas PCR from yesterday. No Gel results.

Vilde

Bought T4 ligase.

William

The N-terminal His tag was made by mixing reverse and forward primers, putting the solution in a 96oC waterbath and left to cool.

Made O/N of TOP10 and NCM17.

Made O/N of double terminator in pSB1C3 (too much medium compared to bacteria - no growth).

Vilde

3A assembly. Followed protocol from iGEM.

1. PartA: Promoter RBS, PartB: Sp Invful long, Plasmid: km
2. A: Promoter RBS, B: Sp-invful short, P: km
3. A: Promoter-RBS, B: Signalling peptide, P: Km
4. A: LacZ Beta Stop, B: T1, P: amp
5. A: LacZAlpha Stop, B: T1, P: Amp
6. A: LacZAlpha NS, B: Ehaj1-T1, P: Km
7. A: LacZAlpha NS, B: Ehaj2-T1, P: Km
8. A: LacZBeta NS, B: Ehaj1-T1, P: Km
9. A: LacZBeta Ns, B: Ehaj2-T1, P: Km
10. A: LacZAlpha NS, B: C-term His-T1, P: amp
11. A: LacZBeta NS, B: C-term His- T1, P: amp
12. A: Promoter-RBS, B: AlphaNS, P:km
13. A: promoter-RBS, B: Beta Ns, P: km

William

O/N cultures of TOP10 and NCM17 was diluted and Jack’s protocol was followed in order to make them competent.

Vilde

Made O/N cultures of colonies from yesterday’s 3A assembly. I got colonies on assembly: 1,4,5,6,7,9 and 13.

Vilde

3A assembly. Due to few colonies from yesterday’s assembly, I suspect something wrong with the parts we already got. I will thus make some of them again from scratch by 3A assembly. Followed iGEM’s protocol and procedure. I ligated them all in to the pSB1K3 plasmid from the kit.

1. Part A: C-term his, Part B: Terminator1 (T1)
2. A. C-term his, B. Double Terminator (DT)
3. A: Ehaj1, B: Dt
4. A: Ehaj2, B: DT
5. A: LacZAlpha Stop, B: DT
6. A: LacZBeta Stop, B. Dt
7. A: signaling peptide (SP), B; T1
8. A: SP, B: DT
9. A: Ehaj1, B: T1
10. A: Ehaj2, B: T1

Transformed the ligated products into competent TOP10 E.coli Cells.

William

The digestion of the 3A assembly protocol was done with:

N-His tag, pBad and constitutive promoter (17O) as part A’s (cut with EcoRI and SpeI). LacZ alpha stop, lacZ beta stop and RBS as part B’s (cut with PstI and XbaI). pSB1A3 and pSB1C3 as plasmids (cut with PstI, EcoRI).

Vilde

3A assembly. Tested colonies from 3A assembly from 04.08 and 05.08 by PCR. Used TaKaRa enzyme and followed 50ul setup. Used elongation 4,3 min and annealing 55degrees. The gel showed only one positive colonies. The gel showed a lot of religated plasmids (bonds of 250bp). I troubleshootet the problem and found out that I will start to de phosphorylated the plasmid to prevent relegation.

Vilde

Minipreped O/N cultures from ligation 04.08. Wanted to test them on PCR to be sure that it is nothing wrong with the way we are doing PCR to test colonies. Tested the concentration on nanodrop – all samples had a concentration between 50-80ng/ul and good 260/280 values. However all the samples had low 230/260 values that indicates that there are co-purified contaminants in the sample.

Vilde

Digested the minipreps and run gel on them. Run gel on the samples minipreped. The results showed that only Alpha-NS-Ehaj1 gave us the expected size of fragments in the gel. However for the smallest parts (RBS, T1 etc) we will not expect to see the bond from the part after digestion only the linearized plasmid. We can thus not say if these parts are correct or just re-ligated plasmids.

Vilde

Tested the one positive colony on PCR to be completely sure it is correct. Used gene specific primers (for Ehaj and LacZAlpha) combined with the iGEM VR and VF2 primers to be sure the fusion protein is in the plasmid.

William

Colonies from a plate with growth of LacZ-beta transformed bacteria were put in tubes, mixed with 20 µl water, heated to 96oC for 10 minutes and then centrifuged at 11.000 rpm for 10 minutes.

A PCR was set up in order to amplify the LacZ beta fragment, but there was no result on the gel.

Vilde

Run gel on PCR from yesterday. Run the gel on the PCR from 06.08. I got bonds as expected and can thus say that the part LacZalpha NS – Ehaj1 is in the plasmid.

Vilde, Håkon

I tested more of the 3A assembly colonies from 05.08 on PCR. Hopefully not all of them are re-ligated plasmids. Used TaKaRa setup (25ul). I tested 3 different colonies from CtermHis –T1 and three from C-termHis-DT. Run the PCR with Håkon.

Vilde

Negative PCR results. Found out that Håkon had used a too short elongation time for my samples to be amplified. Have to do a new PCR on the colonies.

Vilde

Dephosphorylated pSB1C3. Used the pSB1CR Izadora cut (with EcoRI and PstI)and purified from gel yesterday. Used 17ul of purified pSB1C3, 2ul of 10X buffer and 1ul of phosphatase. Let the reaction in 37 degrees for 15 minutes and heat inactivated the enzymes by leaving the sample at 75degrees for 5 minutes. Stored the sample at -20 degrees.

Vilde

Assembled parts into dephosphorylated pSB1C3. Used the dephosphorylated pSB1C3 and parts digested 01.08. Ligation reaction sample: 2ul pSB1C3, 2ul Part, 1ul 10X buffer, 0,5uL enzyme and 4,5ul DH2O. Transformed 5ul and plated out on CAM plates. These parts will be the new biobrikcs we will send to the iGEM register.

William

The N-terminal His tag was made by mixing reverse and forward primers, putting the solution in a 96oC waterbath and left to cool (it did not work the last time(4.8.14)).

The LacZ (19E) and the LacZ (9B) was attempted amplified using PCR. Did not work this time either.

William

DNA concentrations was measured using NanoDrop:

• LacZ beta non stop - 15.2 ng/µl
• LacZ beta stop - 19.0 ng/µl
• EhajI - 15.9 ng/µl
• EhajII - 22.9 ng/µl
• Signal peptide - 56.2 ng/µl

A ligation was set up using all the parts above cut with EcoRI and PstI.

The plasmid used was pSB1C3. Followed the T4 ligase protocol.

The ligation mixtures was mixed with TOP10 competent cells for transformation (Jack’s protocol).

The transformation mixtures was plated onto chloramphenicol plates and incubated until the next day (16h).

Vilde

PCR on colonies. Tested LacZ Beta Non-stop, LacZ-bet Stop and the autotransporter Ehaj1 all in pSB1c3 plasmid backbone. Used VF and VR2 primers. Used Q5 polymerase and followed protocol supplied with the enzyme. Bands of around 500bp were observed for all three samples. This may be religated plasmid backbone.

Vilde

O/N cultures. Made Overnight cultures from the colonies tested even if the PCR did not show expected results. I will try to do another PCR and/Or sequence the isolated plasmid DNA to check if it is religated plasmid I observe.

Vilde

Made new LB Cam medium

Vilde

Made O/N culture of LacZ-gene from iGEM kit that I will use to make constructs.

Vilde

Minipreped the bacterial O/N culture containing LacZ-Gene from iGEM kit.

Vilde

It seems like we have a problem with the digestion of our constructs and or plasmid since we get religated plasmids. I tested all the restriction enzymes stepwise with the corresponding buffer. I used the protocol and setup supplied with the enzyme. I used the EcoRI buffer for EcoRI enzyme, NEB3.1 for SpeI and Cutsmart for XbaI and PstI. I cut a known plasmid from the iGEM kit. Analysis of cut constructs in a gel shows that all restriction enzymes are functional, but has to be done with correct buffer.

I cut the linear plasmid backbone in a stepwise matter

Vilde

Cut pSB1C3. Cut one sample of the plasmid backbone with EcoRI and one with PstI. Used correct buffer for both enzymes. I cut the samples at 37 degrees for 1 hour.

Vilde

PCR cleaned up of the cur plasmid backbone samples eluted the samples in 20ul dH2O.

Vilde

Cut the plasmids backbone with the other enzyme using supplied protocol and buffer. Stored the stepwise cut plasmid backbone in -20 freezer for usage in ligation later.