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.

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).

William

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

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).

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.

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).