Team:Uppsala-Sweden/Notebook

Week 1

This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See protocol SOB- medium, LB medium and Competent cell preparation final). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C (See Competent cell preparation final). We received the pTJ122 plasmid carrying the ccaS, ccaR and cph8 genes as well as the PcpcG2 promoter from Christopher A Voigt at University of California San Francisco.

Week 2

2011-06-27

This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for transforming TOP10 competent cells. The result was good, we measured a competence efficiency of 1.7 * 10^8 transformants /ug DNA.

Blue/green output

The strains carrying the amilGFP (green/yellow output) and amilCP (blue output) plasmids were provided by J.F Miller, UCLA. They arrived on plates witch were malhandled during the delivery. Both colors looked really nice but since the colonies were all mixed into each other we started by re-plating to obtain single colonies.

2011-06-28

Blue/green output

Started overnight cultures of E coli carrying the plasmids pGEM11- amilGFP and pGEM14- amilCP. We followed protocol Overnight culture and glycerol stock for the initial preparations on the output modules.

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells): pSB1A3-J04450 (ampR backbone) – Lei & Sibel </br> pSB1C3-J04450 (CmR backbone) – Lei & Sibel </br> pSB1K3-J04450 (KanR backbone) – Lie & Sibel </br> pSB1AK3-B0014 (Double terminator) –Laura & Pikkei pSB1AK3-B1001 (synthetic terminator) – Karl &Hamid

pSB1A2-B0034 (Standard RBS) - Karl &Hamid pSB2K3-I15008 (ho1, chromophore synthesis gene)- Mohammed, Erik L & Tomas pSB2K3-I15009 (pcyA, chomophore synthesis gene) - Mohammed, Erik L &Tomas pSB1A2-R0011 (PLlacO, lacI repressable promotor) -–Laura & Pekkie pSB2K3-I15010 (cph8 red sensor) – Lei & Sibel

2011-06-29 Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells): pSB2K3-Q03530 (cII inverter) - Tomas, Erik L, Pikkei, Lidaw pSB1AK3-B0015 (Double terminator) - Antonio, Lei, Sibel, Cherno pSB2K3-Q01511 (cI inverter) – Laura & Hamid pSB1A2-R0082 (PompC) - Tomas, Erik L, Pikkei, Lidaw pSB1A2-K093005 (RBS + RFP, red dye) - Laura & Hamid

Streaked on plate pSB3T5-J04450 (low copy vector tet, ori P15A) – From Erik G

Evaluation: All samples were good. pSB1K3 plates with RFP inserts are less red than pSB1A3 and pSB1C3. Re-streaking of the successful transformations from 2011-07-28. The same people who did the transformation proceeded with re-streaking. Cph8 had no colonies at all, neither on the 1x or 10X dilution plate. Apparently there seems to be a problem with cph8. Thus, cph8 (red sensor) had to be redone due to failure in the first attempt.

2011-06-30

Re-streaking of the transformants from 11-06-29 and started overnight cultures from the re-streaked plates from the previous day (11-06-28).

Evaluation: Most transformations succeeded. However, c1inverter gave very little amount of colonies and cph8 failed again. It’s time to use another approach. We got one single colony on the transformation plates and we will screen that one, but if that colony is wrong will try to run a PCR using the standard VF2 and VR primers using the DNA in the kit as template. If there is any BioBrick plasmid in that well, we should get a PCR product. If this will not work out, we need to either synthesize the gene or to PCR up the gene from the Voigt plasmid pJT122. We will then need to clone it into a BioBrick plasmid and run site-directed mutagenesis on it to eliminate the illegal PstI restriction site. Since cph8 is an important part of our project we have to solve this dilemma.

2011-07-01

Started by doing overnight cultures from the re-streaked plates from 11-06-30. After that Lei and Sibel sterile filtered 20 % glycerol for making glycerol stocks. All the overnight cultures prepared 11-06-30 were frozen in -80°C.

2011-07-02

All the overnight cultures prepared 11-07-01 were mixed with glycerol and frozen in -80°C. At this stage content with the work done this far.

Week 3

2011-07-04

Started by doing overnight cultures (3 ml in selective LB) of the strains carrying these plasmids: pSB1A3-J04450 (vector, ampR) pSB1C3-J04450 (vector, CmR) pSB1K3-J04450 (vector, KanR) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB2K3-Q03530 (cII inverter) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC)

The purpose was to make plasmid preparation the day after.

2011-07-05

Green sensor The day started of by running overhang PCR on the DNA template of the green light sensor, which includes the two genes ccaR, ccaS and the PcpcG2 promotor (see protocol ccaR BioBrick, protocol ccaS BioBrick & protocol PcpcG2 BioBrick). Voigt1 (UCSF) provided the green sensor, the red sensor and PcpcG2 on plasmid pJT122 (11,088 bp).

Green and blue output Site-specific mutagenesis was performed on amilGFP and amilCP in order to remove illegal EcoRI sites within the genes (protocol amilGFP_EcoRI, protocol amilCP_EcoRI).

After lunch we did a plasmid preparation of the overnight cultures from 2011-07-04 (purification protocol).

Red sensor At the end of the day we performed colony PCR to verify the length of the single clone we got of red light sensor (cph8) strain.

2011-07-06

Preparation of agarose gel (1%) for the gel electrophoresis. Then we ran our PCR products from 11-07-05 on the gel.

Finally we initiated the first BioBrick assembly. The following entities were assembled:

Chromophore RBS (B0034) + pcyA (I15009) - Lidaw, Pikkei & Johanna RBS (B0034) +ho1 (I15008 ) - Erik L & Tomas

Red output PompC (R0082) + Inv.cl (Q01511) – Kalle, Hamid, Ismael

The plasmids were cut, the enzymes heat inactivated. After mixing the upstream parts, the downstream parts and the backbones, T4 ligase was added and the ligation mixes were left over night in room temperature.

Evalutation: We couldn’t observe any band on the gel corresponding to cph8. Thus the transformation of cph8 has failed completely. We came up with the following conclusions: either there were no or little DNA in the iGEM kit, or the kit just contained something else.

All the other gels were successful but the band for pcpcG2 was slightly weak. The gel bands for ccaR and ccaS looked good. Hence, we have successfully PCR amplified the green sensor parts. Also, the PCR products from the site directed mutagenesis of amilCP_EcoRI and amilGFP_EcoRI showed strong and clear bands at the expected size. Furthermore we can now proceed on cloning ccaR, ccaS and the PcpcG2.

2011-07-07

Re-circularized of pigment vectors after site directed mutagenesis by Sibel. The PCR product was purified, phosphorylated with PNK, template plasmid was degraded using DpnI and finally the linear plasmid was re-circularized using T4 ligase (PCR purification protocol, Phosphorylation of DNA, DpnI digestion protocol). Tomas and Antonio performed transformation of assembly RBS-ho1 (Protocol for transforming TOP10 competent cells). Transformation of mutagenized amilCP_EcoRI and amilGFP_EcoRI by Hamid and Lei.

Digestion of ccaS and ccaR PCR products and plasmid backbone plasmid pSB1C3 by Tomas and Antonio followed up by cloning and transformation by Erik L (BioBrick Assembly Manual). Lidaw And Johanna transformed the RBS-PcyA assembly.

2011-07-08

The transformation from yesterday was successful; hence the same people could proceed with restreaking the transformants. Hamid and Tomas re-did PCR on pcpcG2 using primers with BioBrick overhangs because of the weak band we got last time. This time we got a stronger band.

λ-red

After internal discussions concerning progresses this far, we decided to start preparing the λ-red assembly parallel with the other assemblies. We need to knock out the gene envZ in our E coli strain to avoid cross-talk with our red sensor cph8. To do this, we will use Lambda Red recombineering (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Datsenko and Wanner, 2000).

First step was to perform colony PCR of a strain carrying the plasmid pKD4 (Datsenko) to generate a PCR fragment with a kanamycin resistance cassette flanked by FRT sites and homologies to envZ: “EnvZ- Knock out FRT-Kan” (PCR protocol of envZ knockout FRT-Kan_FRT). We could observe the bands around 1477 bp as we expected. However, we also got a stronger band for the negative control (NC). Even though it seems like we got the right product, the appearance of a band in the NC force us to redo the experiment. Just as a precautious measure.

2011-07-10

Today we screened the transformants from 11-07-08 (3A assemblies: RBS-pcyA, RBS-ho1 and PompC-Inv.cl. Clonings: ccaS and ccaR. Mutagenesis: amilCP and amilGFP. There were 6 clones of each transformants, 42 samples in total.

We selected and suspended one colony from each re-streaked clone in 20-30 µl of PBS. We ran colony PCR from of each suspended colony using taq polymerase using the VF2 and VR primers. We also started overnight cultures in selective medium from the suspensions, 1 ml in each.

As a final attempt to get the cph8 gene from the iGEM kit, we tried running a PCR using the VF2 and VR primers and DNA from the kit as template. To be able to get high quality on the PCR product, we used Phusion DNA polymerase.

Last thing was to run an agarose gel of the PCR products. We also ran the PCR product of the envZ Knockout cassette and cph8 on agarose gel. The envZ Knockout had a clear band (we lowered the annealing temperature from 60 0C to 59 0C). Last thing we did was doing PCR purification of envZ Knockout (Purification protocol). The cph8 showed no bands on the gel, indicating that there simply is no BioBrick plasmid DNA in the kit well.

Week 4

2011-07-11

Today we did a frozen stock of all overnight cultures in -80°C freezer (ccaS clone 1,RBS-pcyA, amilGFP, amilCP, ccaR, RBS-ho1, PompC-Inv.cI). We also started 3 ml cultures by inoculating them with 100 µl of the overnight cultures of the two clones of ccaR, RBS-pcyA and RBS-ho1, the constructs needed for the upcoming assemblies. When the cultures had grown for about 4 hours, we did plasmid preparations of them. We did PCR of amilCP using the pGEM14-amilCP_EcRI plasmid as template and using primers to add BioBrick prefix and suffix. Mutagensis of amilGFP to remove PstI site and ccaS_EcoRI mutagenesis, round one.

After studying the experience section on the Registry’s homepage, we decided to change inverters to parts Q04400 (TetR inverter) and Q04510 (cI inverter). The inverters we originally chose were not so well characterized, and the promoters they used as output might be too weak for our system.

Transformation of parts Q04400 (TetR inverter) and Q04510 (cI inverter).

2011-07-12

The day started by running a gel of the mutagenized ccaS, amilCP-BB10 PCR product, and amilGFP_PstI linear plasmid. At the same time we prepared the samples (ccaR, ccaS, RBS-ho1 and RBS-pcyA) for sequencing. Plating of strains carrying plasmids with terminator B1001, PLlacO, pSB3T5 and pSB4K5. Purification of PCR products of ccaS_EcoRI, amilGFP_PstI, amilCP_BB10, and PcpcG2. Re-ligation of mutagenized ccaS and amilGFP, and cloning of amilCP_BB10.

The transformation of inverters from yesterday didn’t go well. The lambda-cI-inverter had only a few colonies, while the tetR inverter had no colonies at all. So TetR inverter has to be redone. This time the transformation used 2 µl of DNA from the MIT distribution. Re-streak of cI inverter transformants from 2011-07-11.

2011-07-13

We started the day by running EnvZ-KO, ccaS, EcoRI- digested amilCP and amilGFP on a gel. We obtained good results; there were no distinguishable multiple bands on the gel, although the bands seemed a bit smeared. Then we did transformation of ccaS_EcoRI, amilGFP_PstI, pSB1A3-amilCP, pSB-pcpcG2 and pSB4A5 backbone. Re-streak (There were one colony on the whole plate) and colony PCR of tetR inverter (DNA taken from well distributed from MIT).

Started overnight cultures of B1001 terminator, PlacO, PSB3T5 and PSB4K5-backbone (prepared for frozen stock + plasmid preparation) and finally RFP with RBS and lambda c1.inv (prepared for frozen stock + plasmid preparation).

The last thing we did was to make new plates of all the three antibiotics (A, C, K). We also made some plate with tetracycline.

2011-07-14

Re-streak pSB1C3-ccaS_EcoRI, pGEM-amilGFP and pSB4A5. We were supposed to re-streak pSB1A3-amilCP and pSB1A3-PcpcG2 as well, but the transformants had no colonies at all. Suspect wrong plasmid backbone or no ligase added during the cloning attempt. To investigate this we did a transformation of the plasmid backbone (PSB1A3) and plated it on plates containing antibiotics ampicillin, chloramphenicol and kanamycin. We did plasmid prep of pSB1AK3-B1001, pSB1A2-R0011, pSB3T5-backbone, pSB4K5 backbone. New agar plates with antibiotics ampicillin, chloramphenicol and kanamycin, just made in the morning. Assembly of chromophores, step Cloning of tetR inverter into new backbone. Cloning of pSB1A3-amilCP and pSB1A3-PcpcG2 again. Overnight culture of pSB1A3 from the strain collection to test whether it’s right.

The sequencing result arrived today around lunch time! RBS-pcyA strains we sent for sequencing were good. ccaR clone number 2 is better than clone number 1. Clone 1 of RBS-ho1 will be used in further assembly steps.