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Figure 1 Different gBlocks were cloned into pJET and transformed into E. coli TOP10. To verify correct
cloning the plasmids were digested to analyse proper insert size. EsaRv2 = pJET_EsaRv2 cut with
HindIII/SacI, expected insert size 863 bp; cepRv2 = pJET_cepRv2 cut with EcoRI/EcoRV, expected
insert size 944 bp; EsaR = pJET_EsaR cut EcoRI/EcoRV, expected insert size 960 bp. The marked
samples (green arrows) were sent for sequencing.

Figure 2 Different gBlocks were cloned into pJET and transformed into E. coli TOP10. To verify correct
cloning the plasmids were digested to analyse proper insert size. EsaI = pJET_EsaI cut with
BamHI/PstI, expected insert size 765 bp; CFP = pJET_CFP cut with BamHI/HindIII, expected insert
size 934 bp; RFP = pJET_RFP cut SacI/BamHI, expected insert size 1104 bp. EsaRv2 = pJET_EsaRv2
cut with HindIII/SacI, expected insert size 863 bp The marked samples (green arrows) were sent for
sequencing.

Figure 3 Different gBlocks were cloned into pJET and transformed into E. coli TOP10. To verify correct
cloning the plasmids were digested to analyse proper insert size. bla = pJET_bla cut with EcoRV/SpeI,
expected insert size 1083 bp; cepR = pJET_cepR cut with SacI/HindIII, expected insert size 843 bp;
EsaI = pJET_EsaI cut BamHI/PstI, expected insert size 765 bp. The marked samples (green arrows)
were sent for sequencing.

• This week we blunt end ligated eight of our gBlocks (EsaR, EsaR v2, CepR, CepR v2, EsaI, CFP, mRFP, Bla) into pJET 2.1 respectively, transformed them into E. coli TOP 10 and plated some of the obtained transfomants on LB Amp for plasmid preps.
• Those plasmid preps were cut with the corresponding restriction enzymes and an analyzing gel (Fig. 1-3) was run to check for correct size of the inserts.
• Correct plasmid constructs (marked with a green arrow) were sent for sequencing.
• The remaining plasmid DNA of the 11 correct vectors was cut and purified to be used for future OE-PCR

Modeling

• Modelling templates were searched and looked into to know how to start.
• Basic structure of genetic setup was modelled using Matlab with Simbiology.
• Right values for parameters were researched.

Figure 4 Basic setup of genetic interactions.

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• Sequencing results of week 1 were evaluated. For all gBlocks, except for EsaRv2 correct constructs were found.
• Plasmid preps of the correct gBlocks were digested and inserts of the correct size were cut out of the analyzing gel and purified.
• New ligations of EsaR and EsaRv2 with pJET 1.2 were performed and transformed into E.coli TOP 10. Transformants were streaked out again. After plasmid isolation they were again digested to check for the correct size of the inserts. Plasmid preps containing the correct size were sent for sequencing. All of the obtained sequences showed mutations or deletions (Fig 1).

Figure 1 Part of a sequencing result of EsaRv2. The sequence contains a deletion at position 618. As the mutation is in the coding region of the
gene the corresponding sequence could not be used for further work.

• Thus the experiment was repeated.
• pPIC9 was digested with BglII to cut out the E.coli origin of replication as well as the Amp-resistance marker. The linearized vector was further dephosphorylated and purified.
• OE-PCR with of bla_p15A was performed. As the first attempts resulted in some unspecific bands on the analyzing gel (Fig) , the PCR was repeated with more restrictive conditions. Even though an unspecific band remained, a band of the correct size (~2 kb) was obtained and purified. The fragment was digested with BglII to allow cloning into pPIC9.
• bla_p15A was cloned into pPIC9. The resulting circular vector was transformed into E.coli TOP 10 and plated on LB-Amp. However no transformants were obtained, putatively due to the very low concentration of the ligation product. Transformation was repeated with an higher amount of the ligation.

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• After sequencing multiple clones of pJET_EsaR and pJET_EsaRv2 that all contained different mutations we decided to take one clone of each construct that only contained a single deletion and go on with the experiment and cut out the sequenced gBlocks with the corresponding restriction enzymes.
• OE-PCR with the digested and purified fragments of EsaR, CepR and EsaI as well as with EsaRv2, CepRv2 and EsaI were performed to obtain the two fragments EsaR_CepR_EsaI and CepR_EsaR_EsaI_v2, that basically contain the same genetic information, however in a shuffled order. The fused gene fragments was afterwards blunt-end cloned into pJET 1.2. Correct cloning was verified by a restriction digest and an analyzing gel (Fig.).
• As both EsaR_CepR_EsaI as well as CepR_EsaR_EsaI_v2 still contained EsaR-sequences with deletions Quikchange PCR with a primer-pair for each construct was performed to correct the sequence at the respective positions. After the PCR the methylated template plasmids were digested with DpnI to minimize the background, still containing the deletions. After digestions, the corrected vectors were transformed into E. coli Top10.
• Last weeks OE-PCR of bla_p15A was repeated to obtain more of the fragment. The PCR product was purified and digested with BglII. The gene fragment with the sticky ends was ligated again into pPIC9, that had been prepared last week. After ligation the circular vector was transformed into E. coli Top10.
• This week pPIC9_p15A_bla transformation resulted in way more transformants. Plasmid isolation of several constructs was performed. To check for the correct insert the vector was digested with SacI and BamHI (Fig.1) . Correct vectors resulted in ~5600 bp and a ~2000 bp band. One clone was found and used for further experiments.

Figure 1 Analyzing gel of digested vector constructs; Overlap-extension PCR products were blunt-end cloned into pJET 1.2 and transformed into E.coli Top 10. To verify correct cloning, the plasmids were digested to analyse proper size. Std.: Quick-Load Purple 2-Log DNA Ladder; v1: pJET_EsaR_CepR_EsaI_v1 cut EcoRI/SacI, expected insert size ~ 1800 bp; v2: pJET_CepR_EsaR_EsaI_v2 cut EcoRI/SacI, expected insert size ~ 1800 bp; pPIC9: pPIC9_p15A_bla cut PstI, expected insert size ~ 2000bp

Modeling

• Bacterial growth was added to the model by assuming a division time of 45 min.
• Regulation details in the model were adjusted slightly to get a more realistic genetic setup.
• First thoughts were put into how to include interaction with the environment (cell division, diffusion) and will be continued next week.

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• Finally our last gBlock CepI arrived. We put it into the pJET vector with blunt-end ligation. It was then transformed into E. coli TOP10 for plasmid amplification.
• The 2 resulting clones (M1 and M2) were plated for plasmid preps.
• The obtained DNA was cut for a control gel using PstI and NotI. The bands fit the expected size of around 900 bp (Figure 1) and were sent for sequencing.

Figure 1 pJET_CepI (M1 and M2) cut with
PstI and NotI on the control gel.

• The sequencing results showed that one of clones (M1) contained no mutations.
• The remaining plasmid prep of M1 was cut with NotI and SpeI for overlap extension PCR (OE-PCR) and purified with a prep gel.
• That CepI was then used for OE-PCR with CFP. The PCR product was run on a gel. It showed the expected bands at 2000 bp. The DNA was extracted from the gel slices and ligated into pJET. The resulting plasmid was used for transformation into E. coli TOP10.
• This week we set up our controls as well. We transformed pSB3C5_BBa_J04450 (mRFP)pSB1C3_BBa_E0020 (CFP) from the distribution kit 2015 into E.coli Top10 and isolated the plasmids. To get the same vector background with the same origin of replication (p15A)as our pCERI-vector, we cut both constructs with EcoRI/PstI. The cut pSB3C5 was also dephosphorylated. BBa_E0020 was cloned into pSB3C5 afterwards.
• The size verified pPIC9_p15A_bla clone was streaked out again to isolate more plasmid. After plasmid isolation the vector was cut with EcoRV and XbaI to get the p15A_bla fragment for the Gibson assembly of pCERI.