Difference between revisions of "Team:Lanzhou/Testing"

Lanzhou for BIT-China

The LANZHOU iGEM 2016 team consists of 1 graduate students, 14 undergraduate students and 6 instructors. The main part of our project was finished by students from LANZHOU iGEM. We would like to thank our 6 instructors sincerely, for their time and efforts of helping us improve our project. Our work can be divided into 7 parts which are shown below. Each part of work was finished by several student members.

We helped them finish the “another inhibitor” system (arac+pBAD+B0034+TetR+B0015+pTet+B0034+RFP+B0015).(Fig 1)

Fig 1 Inhibitor system construction.

In this system, pBAD is one of the inducible promoter (from BBa_K808000) which can be induced by arabinose. B0034 and B0032 are two kinds of strong ribosome binding sites (RBS) and the strength of B0032 is only 33.96% of B0034. pTet promoter is constitutively ON and repressed by TetR repressor.

We were very glad to help them because this “inhibitor” system is vital for their project. They use the concentration of inhibitor proteins as a signal indicating the plasmid numbers, and it can trigger the downstream reaction by activating the killer gene while the plasmid concentration reduce to the threshold.

There are three crucial components, INP, GFP, and Metal Catcher (Fig 2). INP (Ice Nucleation Protein) is an extrinsic membrane protein of E.coli and acts as an anchor. GFP acts as a reporter while metal catcher can bind heavy-metal ions. This is how we assemble the crucial elements.

At the same time, they helped us finish the pre experimental the fish experiment.

Fig 2 The construction of membrane protein working as anchor.

It is indispensable to investigate whether the surface-displayed E. coli is more potent for detoxification of mercury than common bacteria.

They found that the concentration of mercury ions should be kept at 0.1mg/l. Higher than the value, the fish would be death, lower than the value, the mercury content difference in fish is not obvious.

Fig 3 The survival fish under different suppress of heavy metal ions.

After their help, we identified our subsequent experimental program. (Fig 3)

XM Uni for Lanzhou

The cooperation with Xiamen University has begun since the summer vocation. Working as a very kind and helpful team, they asked helping us in doing TEM to confirm whether the modified protein can be seen on the surface of the bacteria when they knew that we wanted to know whether the different treatment to the bacteria can influence the TEM figure.

They culture our modified bacteria and the control BL21 in the 50mL LB medium adding with 100ug/mL AMP. When the OD600=0.6, add 10ul 0.5mol/L IPTG in IC (Fig 1F). Culture in 16℃ for a night. Before adding ions in the bacteria, wash the bacteria with water. Then add Cu2+ in H-2 to the final concentration 25mg/L (Fig 1E), add Hg2+ to the other group to the final concentration 12.5mg/L (Fig 1C and D). Separate the control in two group and one of it add the same amount of ion in it (Fig 1A and B). After cultured for another 1.5h, observe them with the TEM.

Fig 1 The bacteria under TEM. (A)BL21(control) + Cu²⁺+Hg²⁺ (B)BL21 (C)IC + Hg²⁺ (D)CC + Hg²⁺ (E)IH + Cu²⁺(F)IC + IPTG + Hg²⁺

Unfortunately, compared with before, although the new method of treatment didn’t perform well, we still show our faithful gratitude to them.

n return, we helped them build up a part consisted of RBS lacI and CFP (Fig 2). It is universally acknowledge that LacI is the regulator gene of lac operon in E. coli., which will inhibit the lac operon when expressed. LacI can bind to lactose and it cannot inhibit lac operon under excess lactose, then the gene in lac operon like lacZ、lacY、lacA can be expressed. The expressing level of lacI can be easily detected after connecting CFP with lacI. So we can figure out whether lacI is related to the expressing of other gene conveniently by this improved part.

Fig 2 The construction of their part.