Lab Protocol #1: Making Agar Plate, LB Broth

Making Agar Plate
20g LB Broth
1L H2O
15g Bacto Agar
Mix well first. Pour the mixture into a 2L flask.

Some bacto agar will not be completely dissolved. Therefore, you can always let the bacto agar to settle in your 2L flask, then take the top phase of liquid to flush the remaining bacto agar out.

After that, put it into autoclave machine (wet cycle: this is often preset. But usually it is 100C for 20-30 minutes)

If you wish to make plates for penicillin, wait until the flask to cool down a bit, don’t wait for too long, otherwise the agar will solidify (if you can bear the temperature with your bare skin, the temperature is good). At this time, add penicillin at 1:1000 dilution.

Making LB Broth

40 g LB Broth

2L H2O

Mix well, pour into 4 glass bottles, then autoclave

*When autoclaving liquid in glass bottles, make sure to not tighten the glass bottle completely.

SPOTLIGHT ARTICLES: Control of allosteric signaling switch (Dueber et al. 2003) REVIEW

Today I will briefly discuss one of the landmark papers in protein dynamics. This paper is called Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination, by John Dueber and Wendell Lim from UCSF, published in Science in 2003.

Allosteric signaling switch has been a focus in synthetic biology, as proteins that belong to the signaling pathways are often regulated by allosteric gating mechanisms. Some of these mechanisms are governed by simple binding domains, in which the switch is under autoinhibited state when proper ligand is absent. In this paper, Dueber and his group proposed that combinations of domains could give rise to a diversified number of gating behaviours in response to nonphysiological inputs. This story is highly significant because their results indicate that simple catalytic and interaction domains within a single polypeptide could give rise to different proteins of complex properties. It also advances our knowledge to how proteins of complex functions could be derived.

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To approach the hypothesis, Dueber had developed two key major experimental procedures: (i) switch proteins; and (ii) actin polymerization assays: pyrene-labeled and carboxylated polystyrene bead. There were three types of switch proteins developed: single heterologous ligand; chimeric switch; and heterlogous switch. To develop switch proteins, the ligand motifs and the linker region were PCR-amplified from existing plasmids. These proteins were then expressed with an affinity tag (either 6xHis tag or GST tag), followed by purification and tag removal by Ni beads and TEV protease or glutathione agarose resin. Next, actin polymerization assays were performed. In the case of pyrene-labeled actin assays, pyrene-labeled actin was incubated with Arp2/3, switch proteins and ligand proteins to initiate polymerization. Then the rate of actin polymerization could be measured by fluorescene measurement. Next, bead actin polymerization assay was performed. Carboxylated polystyrene beads were coated with GST fusions to input ligands and PDZ ligand. After incubation, the beads were washed and incubated with Xenopus oocyte extractsand rhodamine-labeled actin, followed by microscopic analysis on rhodamine.

Dueber developed a signaling switch gated by single ligand. This switch was consisted of N-WASP output domain, GBD and B-motif. At the autoinhibited state, GBD and B motif interacted with output and Arp2/3 to suppress output to interact with actin filament. To initiate actin polymerization, Cdc42 and PIP2 bind to GBD and B motif respectively to relieve autoinhibition. This model was consistent with the observation that increasing PDZ ligands could activate the switch. For the second designs, they developed AND-gate switches by linking two domains with an output. There were two classes of designs developed: chimeric and heterlogous switches. Chimeric switch design was regulated by PDZ ligand and Cdc42. The modular domains of this particular design were PDZ and GBD domains, in which Cdc42 could disrupt interaction of GBD and output. Heterologous switch was regulated by PDZ and SH3 ligands. The modular domains were composed of PDZ and SH3 domains. Both classes of switches were subdivided into behavioural classes. Out of 34 switches, 2 showed antagonistic gating, 2 showed OR gate behaviour, and 5 showed AND gate behaviour. From analyzing the behaviours, the basic design principle was identified, in which the affinity of autoinhibition was correlated with the basal repression and input sensitivity. This principle was supported by 2 observations: (1) reducing the PDZ ligand-affinity for switch C11, the switch then resembled AND gate; and (2) increasing PDZ ligand affinity turned H15 switch from behaving like OR gate to AND gate. Their results also indicated the importance of linker region to switch behaviour, as the increase of linker region from 5 to 20 residues could increase the sensitivity of inputs. Furthermore, in the case of antagonistic switches, PDZ ligand was found to act as an activator while SH3 increased the basal level of repression. PDZ was responsible for the autoinhibitory interaction with output, and SH3 interaction was responsible for destabilizing the PDZ interaction. In the case of positive integrating switches, both domains worked together to downregulate the output activity. Interestingly, disruption of both was required to activate the output function.

Dueber and his group have effectively addressed the main question. The results from simple binding domains and corresponding ligands had supported the notion that complex signaling circuits could be derived from simple domains, and more importantly, domain recombination could potentially derive proteins with novel regulated functions. For future direction, the geometry of the output domain must be investigated. In order for the output domain to be regulated by other domains and to carry its native function, certain geometry must be preserved. To understand the preservation, X-ray crystallography and mass spectroscopy should be conducted on samples: output domain bound to GBD and output domain bound to actin filament.

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Reference: Dueber JE, Yeh BJ, Chak K, Lim WA. Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination. Science. 2003 Sep 26;301(5641):1904-8.

Spotlight articles: Trading Places on DNA (Yuzhakov et al. 1999) Review

Today I will briefly discuss one of the landmark papers in molecular biology. This paper is by Alexander Yuzhakov, Zvi Kelman and Mike O’Donnell, from the Rockefeller University and the HHMI in New York City.

DNA duplex has an elegant structure, yet its replication relies on a sophisticated and complex network of factors. During DNA replication, helicase unwinds the DNA duplex. SSB binds to this single stranded region to maintain and stabilize the structure. On the lagging strand, primase is required to produce a series of RNA primers. The β sliding clamp is then loaded onto the primed site to enable the extension by DNA polymerase III. This landmark paper, by O’Donnell group, tries to understand how primase is displaced from DNA. They hypothesize that there is a component in the DNA replication that removes primase after RNA primer is made. This paper is highly significant as (i) it shows that there is a competition between components to enable efficient and regulated DNA replication; (ii) and due to this competition, components that are present at a low concentration, such as primase, can be recycled.

This paper started from trying to understand the temperature-sensitive replication defect on phage DNA when it is coated with SSB-113 mutant. When phage G4 ssDNA is primed with synthetic RNA, both SSB- and SSB-113-coated ssDNA can replicate at permissive and restrictive temperatures. However, at restrictive temperature, SSB-113-coated ssDNA can not be replicated. This defect is not due to the malfunction of primase. Knowing that the β-clamp is loaded after primase priming, they then continue to see if the loading is defective. Using 32P-labeled β-clamp, they realize that the clamp loading is defective in SSB-113-coated ssDNA at restrictive temperature, which suggests at restrictive temperature, the SSB-113 prohibits the clamp loading, which may consequently leads to replication defect. They then continue to ask what causes the displacement of primase. β-clamp alone does not displace primase, but DNA Pol III and the β-γ complex does. Interestingly, χ subunit of Pol III alone displaces primase effectively. Because Pol III does not displace primase at restrictive temperature when ssDNA is coated with SSB-113, they then hypothesize χ’s interaction with SSB is temperature sensitive. χ does not interact with primase. When ssDNA is coated with SSB-113, χ shows decreased interaction comparing to SSB-coated ssDNA. In contrast, primase does not show this interaction discrepancy with SSB-113. Interestingly, as χ increases, primase bound to the ssDNA decreases, which indicates that there is a competition between χ subunit and primase for SSB interaction. To confirm the competition, they compare the level of synthesized DNA and bound primase in the presence and absence of χ. In the absence of χ, primase remains bound to the ssDNA, and replication of lagging strand is defective. In the presence of χ, primase is displaced, and lagging strand is replicated. Last but not the least, they show that the displacement by Pol III is polymerase-specific, as other polymerases do not replicate. This primase-polymerase switch is also species-specific as human clamp PCNA does not load when phage primase is present. The primase also protects the RNA primer from RNAse. Taken together, primase-polymerase switch depends on the competition between χ subunit of RNAP and primase for interacting with SSB. Primase also protects the RNA primer, and its displacement from SSB-coated ssDNA is species- and polymerase-specific.

Mike O’Donnell’s group has presented an important mechanism in DNA replication with well-supported evidence. The paper’s successful attempt to bring the switch concept in phage G4 ssDNA into a broader and general concept, such as replication fork, is admirable. This brilliant attempt is shown in Fig. 5, with well thought-out designs to prove the displacement function of χ subunit. O’Donnell’s approach to answer the questions is logical, and I believe that this paper has answered the questions posed. However, the author has yet addressed why the primase is not displaced at restrictive temperature. On 154, the author states, “the χ subunit of Pol III holoenzyme interacts much more weakly with SSB-113 than with wild-type SSB”. It is possible that the res. 113 is essential for interacting with χ. And at restrictive temperature, interaction with res. 113 is lost possibly due to conformational change. SSB-113 carries Pro (non-polar) to Ser (polar) mutation. To see if the polarity of this residue is important for interaction with χ, we can mutate the Pro113 to Cys113 (slightly polar). By immobilizing the Cys113 mutant in SPR, we can see if this slightly-polar residue would result in intermediate interaction. Fig. 1A illustrates how the primase produces RNA primer in a 5′-3′ direction. How does χ detect the primase-bound SSB? As χ does not interact with primase (Fig. 4C), does primase binding change the structure of SSB providing a signal for χ subunit recognition? To address this question, we have to first understand where χ binds to in ssDNA. In other words, does χ bind to the opposite or the same face of primase? To approach, we can perform DMS methylation footprinting to probe for MG/mg regions that are protected by χ and primase. By comparing the binding regions, we will have a better understanding on how the displacement works.

 

 

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Reference:

Yuzhakov A., Kelman Z., O’Donnell M. (1999). Trading places on DNA — a three point switch underlies primer handoff from primase to the replicative DNA polymerase. Cell (96, 153-163).