Neon Coli-Necessary Expression Only

Lab Notebook

Week1 20180325-20180331

Moving in our new lab! The first week Huang Tianze, Shen Yunhao, Wang Tingxuan, Zhou Manxuan, Wu Yan, Ge Xiaofei and Zhou Liqun worked on the biobrick part transformations and sequencing. Also we received some help from our PI, Professor Chen’s lab. They provided us with sfGFP and RFP reporters. We prepared the necessary equipment and materials, like LB medium and plates. For the first batch of plates we used agarose instead of agar. The plates turned to stone afterwards hahaha.

Week2 20180401-20180407

The parts we intended to use were amplified and sent for sequencing by Huang Tianze, Shen Yunhao, Li Yibo and Wang Tingxuan. We started the pilot studies for Neon Coli and divided the work. Three groups of people started working on characterization of the quorum sensing system Lux and Las, the red light sensing system and the CRISPRi system.

Week3 20180408-20180414

Still doing transformation…Some parts are just unbelievable. Some sequencing results were inconsistent so we had to find another copy. Huang Tianze, Shen Yunhao, Li Yibo and Wang Tingxuan, Zhou Manxuan, Wu Yan and Zhou Liqun started designing the pilot test plans. We decided to use InFusion ligation technique because it sounds much faster than restriction cloning. (We were so wrong…)

Week4 20180415-20180421

Primers were flying in! Tsinghua iGEM lab is on full capacity. Huang Tianze, Wang Tingxuan, Zhou Manxuan, Wu Yan and Shen Yunhao were doing PCRs and gel recovery, building the pilot plasmids. The Phusion polymerase we were using was quite fickle. We spent half of the time debugging the PCRs.

Week5 20180422-20180428

First batch of InFusion! Huang Tianze and Wang Tingxuan tried to ligate the CRISPRi test plasmids. Zhou Manxuan, Wu Yan and Shen Yunhao repeated the PCRs for light sensing and quorum sensing plasmids

Week6 20180429-20180505

We tested the CRISPRi plasmids but they had no effect on sfGFP expression. They were definitely not right because enzyme cutting revealed inconsistent bands. Retested the recovered PCR products. Shen Yunhao built the first lux QS plasmids. Huang Tianze and Wang Tingxuan decided to order new primers for CRISPRi construction.

Week7 20180506-20180512

Retried CRISPRi InFusion. Huang Tianze and Wang Tingxuan spent loads of time on PCR. Some primers were just too long. Zhou Manxuan and Zhou Liqun finished the light sensing reporter plasmid.

Week8 20180513-20180519

Retried InFusion, still no CRISPRi plasmids…Zhou Manxuan, Zhou Liqun and Wu Yan used restriction enzymes to test the light sensing reporter plasmid OG, the results were inconsistent. Redid PCR and InFusion for OG and the sensor plasmid HPTC. Shen Yunhao transformed the lux reporter plasmid, on LB plates the colonies seemed quite green. Lux pR reporter must be leaky.

Week9 20180520-20180526

Still trying… CRISPRi and light sensing system colony PCRs generated no proper ligated plasmid. Shen Yunhao started building the positive feedback circuit. Dr. Li Peng suggested we use overlapping extension PCR to ligate smaller parts together before InFusion.

Week10 20180527-20180602

Overlapping PCR worked! Huang Tianze and Wang Tingxuan built the strong expressed CRISPRi plasmids that successfully repressed the production of sfGFP. Even though some colonies still leaked. Started to build medium and weak dCas9 generators. Zhou Manxuan and Zhou Liqun also began to use overlapping extension to build HPTC plasmid.

Week11 20180603-20180609

Weaker dCas9 generator showed better repression of sfGFP. M0.3, W1.0, W0.6 and W0.3 CRISPRi plasmids eliminated co-transformed sfGFP generator, while S1.0, S0.6 and S0.3 still showed some leakage. We were very puzzled. Shen Yunhao finished the lux positive feedback plasmid. Even before extraction the broth shone very brightly. We decided to build an IPTG induction device above the quorum sensing system.

Week12 20180610-20180616

Light sensing still had no results. We decided to divide and conquer, separating the sensor HPTC to several parts and then ligate. However sequencing and colony PCRs indicated that the construction of smaller parts were no lighter work than the entire plasmid. We decided to change the construction plan.

Week13 20180617-20180623

Sequencing of the S1.0 dCas9 plasmid revealed multiple mutations. Strong expression of dCas9 may be too toxic to the bacteria that it has to be mutated so that the bacteria can survive. Therefor we decided to use W0.6 dCas9 generator in the following experiments.

Week14 20180624-20180630

Summer holiday (also finals) were creeping in! Nothing really changed. Phusion was still fickle, gel recovery was still lacky and InFusion was still very difficult. We started the PCRs for IPTG inducible quorum sensing devices.

Week15 20180701-20180707

Summer holiday officially starts! We asked around other iGEM teams and found out that nearly everyone had encountered troublesome promotors, leaky, unreliable or otherwise had low intensity. We finally finished light sensing reporter LSP. But even without its sensors the reporter sfGFP still expressed. We reconsidered our position and decided to focus more on expression control instead of artistic presentation. Also we welcomed the high school teams SHSBNU_China and RDFZ_China. They settled into the lab next door.

Week16 20180708-20180714

Wang Tingxuan and Huang Tianze stayed behind… Life was all about PCRs, overlapping PCRs and gel recovery. In order to improve our PCR success rate we ordered Q5 premix. IPTG induced las QS plasmid was constructed. We sprayed some IPTG on the plate but there was no effect. We finished the design of the NEON system.

Week17 20180715-20180721

IPTG induction still had no results. Our advisor Wang Xuan suggested that maybe lacI expression was too strong. Preliminary model simulation indicated that lux pR was a knot where loads of biochemical reactions intersects. We decided to build a less leaky but more sensitive lux pR promotor and started the design of lux pR-HS.

Week18 20180722-20180728

We realized that previous plasmid construction was not efficient, so Huang Tianze designed a new set of plasmid construction plans. The parts are InFusioned into the smallest transcription units and individually amplified, then use 3A assembly to assemble the units together to form the test plasmids. So…even more PCRs and gel recoveries.

Week19 20180729-20180804

The new plans worked out quite well. This week we sequenced the unit plasmids and confirmed most of them. Wang Tingxuan and Chen Xinyi ligated the lux pR test plasmid. Yao Liang designed the lux pR mutants. Started to build Neon and Safety Catch.

Week20 20180805-20180811

Wang Tingxuan went to the regional meet up hosted in Peking University. We sent out the first batch of our HP surveys. Pilot lux pR testing showed that AHL induction intensity was quite high. But IPTG induction of lacI+pI promotor still showed no results. RDFZ_China suggested that we use Tac promotor.

Week21 20180812-20180818

Wang Tingxuan designed new quorum sensing test systems of Rhl and Cin. IPTG induction still generated no results. Started Ptac test plsmid construction. We considered the effect of lacI dosage and decided to use three different levels of induction.

Week22 20180819-20180825

We tested lux pR and mutants AHL induction with Thermo Varioskan Flash. The preliminary results lead us to design the sequence of lux pR-HS. Designing plasmids for IPTG induction of CRISPRi. Ordering more primers hahaha.

Week23 20180826-20180901

Finished construction of Neon plasmids. Positive feedback was indeed very strong, the fluorescent intensity was way stronger than lux pR AHL induction. CRISPRi plasmids were difficult to assemble because dCas9 was too long. We aborted the original plan and decided to build dCas9 and gRNAs into separate transcription units and ligate them later. Also Ge Xiaofei and Chen Xinyi attended CCiC in Shanghai and sent out our questionnaires.

Week24 20180902-20180908

Uploaded our safety form! Finished Neon-HS construction and all the lux pR mutants. Discovered that dCas9 contained an illegal EcoRI site and gRNA contained illegal SpeI sites. Ordered primers for point mutation.

Week25 20180909-20180915

Uploaded the final version of our abstract. Finished construction of dCas9 generators. IPTG induction of Ptac promotors were still unsuccessful. Finished the construction of different levels of lacI generators.

Week26 20180916-20180922

Began building the plasmids for part registry. IPTG induction finally had results. Indeed too much lacI protein made Ptac not respond to IPTG. Lower lacI concentration would suffice. Repeated lux pR mutation analysis.

Week27 20180923-20180929

Finished construction of plasmids for IPTG induced CRISPRi, Safety Catch and Safety Catch-HS. Started lacI dosage analysis. Nearly finished the plasmids for part registry.

Week28 20180930-20181006

Prepared for the exhibition at China Science and Technology Museum. Tested the IPTG induced CRISPRi system. Prepared the test of NEON system.

Week29 20181007-20181013

Sent out the plasmids just in time. While transforming the NEON plaids we discovered that unbalanced copy number can cause variation in the outcome of the circuits. We decided to try to ligate them together.

Week30 20181014-20181017

Working hard on Wiki! We repeated other experiments for more data and continued the NEON characterization.
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